Albumin production and cell proliferation

ABSTRACT

The present invention provides short activating RNA molecules which up-regulate albumin production. The present invention also provides methods of up-regulating albumin production, such methods involving the use of short activating RNA molecules capable of increasing the expression of albumin. The present invention also provides the use of the short activating RNA molecules in therapy, such as treating or preventing a hyperproliferative disorder and/or a disorder characterised by hypoalbuminemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/013,866 filed Feb. 2, 2016 entitled ALBUMIN PRODUCTION AND CELLPROLIFERATION, which is a divisional application of U.S. applicationSer. No. 14/128,147 filed Dec. 20, 2013 entitled ALBUMIN PRODUCTION ANDCELL PROLIFERATION, which is a national phase entry of PCT ApplicationNo. PCT/GB2012/051422 filed Jun. 20, 2012, which claims the benefit ofpriority of U.S. Application No. 61/499,637 filed Jun. 21, 2011, thecontents of which are each incorporated herein by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The sequence listing filed, entitled2058-1007USDIV_SL.txt, was created on Jul. 21, 2017 and is 7,034 bytesin size. The information in electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of up-regulating albuminproduction. The methods involve the use of short RNA molecules capableof increasing the expression of albumin. The invention also relates tothe design and synthesis of such short RNA molecules and their use intherapy, such as treating hypoalbuminemia or cancer. Methods ofinhibiting cell proliferation by up-regulating albumin production arealso provided. Subject matter disclosed herein thus relates generally toshort activating RNA (saRNA) and, in particular, saRNA for impactingcell proliferation.

BACKGROUND OF THE INVENTION

Current methods of up-regulating the expression of a gene of interesttypically require the introduction of extra copies of the gene into acell, either by using viruses to introduce extra copies of the gene intothe host genome or by introducing plasmids that express extra copies ofthe target gene. By contrast, the present invention provides shortactivating RNA molecules which can upregulate gene expression,particularly albumin production.

Various studies indicate that short activating RNA (saRNA) can target apromoter region of a gene and activate the gene. Some have proposed amechanism (Mechanism A) where short double-stranded or single strandedRNAs find a match in a promoter region of a gene to form a complex thatbinds to the promoter region and removes so-called “off” tags; andanother mechanism (Mechanism B) where a cell produces RNA copies of apromoter region that somehow block production of a protein to silence agene; and where short double-stranded or single stranded RNAs find amatch to bind and destroy the RNA copies. These proposed mechanisms(e.g., believed to be one or possibly both) lead to turning “on” atarget gene, for example, to provide for production of a protein. FIG. 1shows approximate graphical diagrams that illustrate Mechanism A 110(see graphics 112 and 114) and Mechanism B 120 (see graphic 122) withrespect to a promoter region, a coding region and a double stranded RNAthat can form a complex (see, e.g., Holmes, B., “Turn genes on, turndiseases off”, New Scientist, 7 Apr. 2007, which is incorporated hereinby reference).

RNA interference (RNAi) is an important gene regulatory mechanism thatcauses sequence-specific down-regulation of target mRNAs. RNAi ismediated by “interfering RNA” (iRNA); an umbrella term which encompassesa variety of short double stranded RNA (dsRNA) molecules which functionin the RNAi process.

Exogenous dsRNA can be processed by the ribonuclease protein Dicer intodouble-stranded fragments of 19 to 25 base pairs, preferably 21-23 basepairs, with several unpaired bases on each 3′ end forming a 3′ overhang.Preferably, each 3′ overhang is 1-3, more preferably 2, nucleotideslong. These short double-stranded fragments are termed small interferingRNAs (siRNAs) and these molecules effect the down-regulation of theexpression of target genes. Since the elucidation of their function,siRNAs have been used as tools to down-regulate specific genes.

A protein complex called the RNA-induced silencing complex (RISC)incorporates one of the siRNA strands and uses this strand as a guide torecognize target mRNAs. Depending on the complementarity between guideRNA and mRNA, RISC then destroys or inhibits translation of the mRNA.Perfect complementarity results in mRNA cleavage and destruction and asresult of the cleavage the mRNA can no longer be translated intoprotein. Partial complementarity—particularly with sites in the mRNA's3′ untranslated region (UTR)—results in translational inhibition.

Recently it has been discovered that although RISC primarily regulatesgenes post transcription, RNAi can also modulate gene transcriptionitself. It is believed that short RNAs regulate transcription bytargeting for destruction transcripts that are sense or antisense to theregulated RNA and which are presumed to be non-coding transcripts.Destruction of these non-coding transcripts through RNA targeting hasdifferent effects on epigenetic regulatory patterns depending on thenature of the RNA target. Destruction of ncRNA targets which are senseto a given mRNA results in transcriptional repression of that mRNA,whereas destruction of ncRNA targets which are antisense to a given mRNAresults in transcriptional activation of that mRNA. By targeting suchantisense transcripts, RNAi can therefore be used to up-regulatespecific genes.

A published US patent application 2010/0210707 A1 ('707 application),which is incorporated by reference herein, sets forth some technologyfor use of saRNA. The '707 application describes selection of anon-coding region of a nucleic acid sequence of a gene to provide forcomplementarity of a saRNA strand to, in turn, provide for an increasein transcription of the corresponding gene. Such an approach infers thatthe “target” is known a priori. Further, the '707 application statesexplicitly “saRNAs do not target cryptic promoter transcription”. Indetail, the '707 application notes that expression of E-cadherin, p21and GAPDH was detected using gene specific primer sets; that no cryptictranscript was detected using primers complementary to the E-cadherinpromoter; and that no cryptic transcript was amplified in the p21promoter.

The '707 application also describes an saRNA molecule with at least afirst ribonucleic acid strand with a 5′ region of complementarity to anon-coding sequence of a gene, where the gene encodes a polypeptide thatinhibits cellular proliferation, and a 3′ terminal region of at leastone nucleotide non-complementary to the non-coding sequence, whereadministering of the saRNA provides for an increase in expression of thepolypeptide and a decrease in cellular proliferation. As explicitlystated, the referred to polypeptide itself “inhibits cellularproliferation”.

As described herein, various technologies, techniques, etc., can providefor saRNA, optionally without specific a priori knowledge, where suchsaRNA may be administered, directly or indirectly, to impact cellproliferation. In various examples, cell proliferation is impacted notby presence of a polypeptide that inhibits cell proliferation but ratherby a controlling a mechanism (or mechanisms) for production of one ormore polypeptides. As described herein, each of such one or morepolypeptides may or may not, by presence of the polypeptide moleculeitself, inhibit cell proliferation.

DETAILED DESCRIPTION

The present inventor has set out to provide a new method of upregulatingalbumin production in vivo or in vitro. He has developed new short RNAswhich up-regulate albumin production. This up-regulation is achieved byup-regulating a target gene involved in albumin production. These RNAsactivate gene expression, so they are also called short activating RNA(saRNA). The terms “short RNA” and “saRNA” are used interchangeablyherein.

Without wishing to be bound by theory, it is believed that the short RNAmolecules of the invention may act through mechanism A and/or Bmentioned above. The short RNAs of the present invention may achievemodulation of the target gene by inducing the siRNA-like cleavage of thetarget RNA transcript which is antisense to a region of the target gene.Short RNAs of the present invention might also be able to act, incomplex with Argonaute proteins, as anchors for regulatorychromatin-modifying proteins. The saRNA mechanism of action may involvechromatin remodelling, for example, through Polycomb group proteins.Polycomb group proteins can apparently directly interact with ncRNAs,including promoter-associated RNAs, and thereby be recruited topromoters and effect silencing. The saRNAs may therefore, by interferingwith such Polycomb-recruiting ncRNAs, reduce Polycomb-levels atpromoters and allow “positive” chromatin remodeling complexes such asTrithorax group proteins to establish positive histone marks.

Thus, the saRNA molecules may up-regulate albumin production viadown-regulation of a target antisense RNA transcript.

The inventor has used an advantageous method/algorithm for theidentification of suitable RNA target transcripts and for the design ofthese saRNAs. The inventor has therefore provided novel short RNAs whichtarget RNA transcripts in the host cell in order to upregulate targetgenes involved in albumin production.

The short RNAs of the invention are also referred to herein as “albuminproduction up-regulating” RNAs. Preferred features of the saRNAs of theinvention are discussed below.

By a “target gene involved in albumin production”, conveniently referredto herein as a “target gene”, is meant a gene which whenactivated/upregulated results in increased albumin production. Albuminproduction may inter alia be upregulated using saRNAs which upregulatethe albumin gene or saRNAs which upregulate the CEBPA gene, as shown inthe Examples. Thus, the target gene may conveniently be the gene whichencodes albumin (the “albumin gene”), in which case the saRNA may besaid to have a direct effect. Alternatively, the target gene may encodea factor which immediately or ultimately regulates the production ofalbumin, in which case the saRNA may be said to have an indirect effect.Such a factor may for example be a transcription factor. Preferredexamples include CEBPA and HNF4alpha.

Albumin is the body's predominant serum-binding protein. It has severalimportant functions. It affects osmotic pressure, so when albumin nolonger sustains sufficient colloid osmotic pressure to counterbalancehydrostatic pressure, oedema develops. Albumin transports varioussubstances, including bilirubin, fatty acids, metals, ions, hormones,and exogenous drugs. It also affects platelet function.

CCAAT/enhancer binding protein alpha (CEBPA, also known as C/EBP alpha)is an intron-less gene which encodes a basic leucine zipper class ofprotein. The CEBPA protein consists of 2 N-terminal transactivationdomains, a basic DNA binding domain and a C-terminal leucine zipperdomain. CEBPA is a sequence specific, DNA binding protein purifiedinitially from rat liver nuclei. The binding sites for CEBPA includeCCAAT boxes and enhancer core homologies. It is capable of selectivelyintegrating with cis-regulatory DNA sequences to positively control mRNAsynthesis. Based on its ability to positively regulate cis-regulatorysequences, CEBPA is classed as a transcription factor.

CEBPA is expressed in a variety of tissues where it plays an importantrole in the differentiation of many cell types including adipocytes,type II alveolar cells and hepatocytes. In the mouse, CEBPA is foundmost abundantly in fat, liver and lung tissue. CEBPA is restrictedfurther to terminally differentiated hepatic parenchymal cells. Thefunctional role of CEBPA in liver cells was reported by Darnell et al.,showing its regulation of alpha-1-antitrypsin, transthyretin andalbumin. Furthermore expression of functional CEBPA in the liver cellline (HepG2) results in increased levels of cytochrome P450, asuperfamily of monooxygenases that participates in the metabolism ofendogenous substrates and plays a key role in detoxification andmetabolic activation of key xenobiotics. CEBPA plays a physiologicallyrelevant role in liver specific genes, as evidenced by its presence onthe promoter element of the albumin gene.

HNF4A (hepatocyte nuclear factor 4 alpha) is a nuclear transcriptionfactor that controls the expression of several genes. It binds as ahomodimer, and may play a role in development of the liver, kidney, andintestines. HNF4A mutations are associated with monogenic autosomaldominant non-insulin-dependent diabetes mellitus type I.

Albumin production upregulation according to the present invention maybe achieved by upregulating a single target gene, preferably selectedfrom the target genes discussed above, or by upregulating at least 2 orat least 3 different target genes. Preferred combinations areupregulation of the albumin gene and the CEBPA gene; albumin gene andHNF4A gene; CEPBA gene and HNF4A gene; or albumin gene, CEBPA gene andHNF4A gene. Upregulation of any particular target gene may be achievedusing a single saRNA (single or double stranded) or a combination of twoor more different saRNAs (single or double stranded). When two or moretarget genes are upregulated, then a combination of at least one saRNAspecific for the first target gene and at least one saRNA specific forthe second target gene are used. Thus, at least 2, 3, 4, 5, 6, 7, 8, 9,10,11, 12, 13 or 14 different saRNAs may be used in any combination.Preferably, the saRNA comprises a first strand comprising or consistingof a sequence of SEQ ID NO: 5-36.

As discussed below, many conditions are characterised byhypoalbuminemia, so there is a need to increase albumin levels inpatients suffering from such conditions. The present invention thereforeprovides new therapeutic approaches for the treatment of suchconditions.

Surprisingly, the inventor has found that up-regulating albuminexpression in a hyperproliferative cell inhibits proliferation of thatcell and up-regulating albumin expression in vivo inhibits tumordevelopment and growth, as shown in the Examples. This opens up newtherapeutic approached for the treatment of hyperproliferativedisorders.

As described herein, a cell may be modified by administration of a shortactivating RNA to thereby cause the cell to increase production of amolecule where production impacts cell proliferation. As an example, ahepatocyte is modified by administration of saRNA to increase productionof albumin. In turn, the ability of the hepatocyte to divide (i.e.,proliferate) is impacted negatively. In other words, the mechanism ormechanisms associated with the increase in production of albumin causetreated hepatocytes to exhibit reduced proliferation. As describedherein, saRNA may be useful for treatment or prevention of cancer, suchas primary liver cancer. For example, as to liver cancer, treatment orprevention may occur by administering saRNA to patients with livercirrhosis (e.g., due to viral hepatitis or ethanol intoxication).

As an example, trials were undertaken with an objective of exploring theeffect of up regulating albumin mRNA transcript on cell proliferation inthe human hepatocellular carcinoma (HepG2) cell line. The trialsincluded use of synthetic saRNA targeted at albumin, which wastransfected into HepG2 using the Nanofectin liposomal method. For thesetrials, successful transfection was measured by an increase in mRNAlevels in HepG2 cells. To demonstrate the impact, change in cellularproliferation was measured using tetrazolium salt,4-[3-(4-iodophenyl)2-(4nitrophenyl)2H-5-tetrazolio]1,3benzenedisulfonate (WST-1) proliferation assay.

These trials provided results that demonstrated that saRNA, targeted atthe promoter region of the albumin gene, was successfully transfectedinto HepG2 cells (e.g., as albumin mRNA levels increased when comparedto cells transfected with a scrambled RNA control). These results showedthat cell proliferation markedly decreased only in cells that werereprogrammed to upregulate albumin expression. Such an effect was alsoobserved in a human cell line expressing albumin. Also noted, for ratliver fibroblasts that do not express albumin, transfection of these ratcells with the same saRNAs, did not result in upregulation of albuminmRNA or a change in cell proliferation.

As described herein, enhancing albumin transcripts in HepG2 cellsindirectly represses cell proliferation. In line with the establishedrole that the transcription factors p53, HNF4-α, CEBPA-α and CEBPA-βhave with albumin expression and induction of apoptosis; techniques,technologies, etc., described herein provide opportunities for targetingof hepatocarcinoma with saRNA molecules specific to albumin. Moregenerally, as described herein, saRNA molecules can increase productionof one or more molecules whereby the mechanism or mechanisms associatedwith production impact cell proliferation, to specifically reduce cellproliferation. Reduction in cell proliferation can be beneficial fortreatment of various conditions.

Hepatocytes are generally perceived as being important for maintenanceof several vital functions. For example, they can regulate carbohydrateand lipid metabolism and detoxification of exogenous and endogenouscompounds. They can also produce plasma proteins such as albumin.Albumin is a typical liver-specific gene which is important for thetransportation of particles through the body and the preservation ofserum colloid osmotic pressure in the blood. The gene for albumin(albumin gene) is highly expressed in the liver after birth, and itsregulation is transcriptionally controlled as indicated by the numerousfactors that bind to the albumin promoter element (see, e.g., Panduro etal., 1987; Tilghman and Belayew, 1982). Several cis-acting elements inthe albumin promoter exists (sites A-F). The B and D sites have beenshown as to being important as liver-specific transcription factors bindto these elements (see, e.g., Maire et al., 1989). HNF-1 has been shownto bind to the B site (see, e.g., Courtois et al., 1988; Lichtsteinerand Schibler, 1989). Members of the C/EBP family which belong to theleucine zipper proteins of transcription factors have been shown tointeract at the D site of the albumin promoter (see, e.g., Descombes etal., 1990; Landschulz et al., 1988; Mueller et al., 1990).

In general, several events may influence expression of the albumin gene.Under physiological conditions, extracellular oncotic pressure has beenshown to controls albumin gene expression via HNF-1 (see, e.g.,Pietrangelo et al., 1992; Pietrangelo and Shafritz, 1994). Duringpathological states, such as the acute phase response of the liver,albumin gene expression has been shown to be down-regulated (see, e.g.,Trautwein et al., 1994). In recent years several transcription factorshave been cloned which have shown involvement in the regulation of theacute phase genes. These include STAT3, C/EBP-β, IL6-DBP, NF-IL6, LAP orCRP2 (see, e.g., Akira et al., 1990; Akira et al., 1994; Cao et al.,1991; Chang et al., 1990; Descombes et al., 1990; Poli et al., 1990;Williams et al., 1991; Zhong et al., 1994). The C/EBP family oftranscription factor is particularly relevant as members of this familybind to the D site of the albumin promoter and also regulates cell cyclearrest via p53 and HNF4-α (see, e.g., Barone et al., 1994; Buck et al.,1994; Johnson, 2005; Nagao et al., 1995). HNF4-α and CEBP-α have beenboth shown to be critical for liver function and differentiation (see,e.g., Hayhurst et al., 2001; Lee et al., 1997; Wang et al., 1995). Somestudies have shown that transcriptional repression of both HNF4-α andCEBP by overexpression of the tumour suppressor p53 correlates with poorliver differentiation in cancer (see, e.g., Itoh et al., 2000; Nagao etal., 1995; Ng et al., 1995). An increase in p53 levels has been shown tocorrelate with a decrease in albumin expression possibly throughinhibition of CEBP-α and β transcriptional activity (see, e.g., Kubickaet al., 1999). In normal conditions, wild type levels of p53 are verylow and are increased only in response to cellular stress such as DNAdamage, withdrawal of growth factors and hypoxia (see, e.g., Prives,1998). Tight regulation of p53 protein level has been shown to berequired to prevent blockage of the cell cycle and induction ofapoptosis.

Albumin is not thought to have a role in cell proliferation, i.e. it isnot involved in the cell cycle and not required for cell proliferation,nor are normal levels of albumin thought to affect cell proliferation.It can be contrasted with proteins such as cyclins and cyclin-dependentkinases which are involved in cell proliferation. Without wishing to bebound by theory, the inventor proposes that by upregulating albuminproduction, the cell's resources are diverted from cell proliferationand cell proliferation consequently slows down or steps entirely.

As described herein, as an example, upregulation of albumin (see FIG. 2)may be “tricking” cells into a reversion back to normal conditions wherep53 levels are reduced—resulting in an increase in transcription ofHNF4α, CEBPA-α and β- to restrict cell proliferation and growth (see,e.g., Maeda et al., 2002). Evidence from the trials demonstrate thatupregulation of albumin by saRNA markedly represses proliferation of aliver cancer cell line (see FIG. 3).

Thus, in one aspect the present invention provides a method of treatingor preventing a hyperproliferative disorder, comprising administering toa subject in need thereof a short activating RNA which up-regulatesalbumin production and thereby inhibits cell proliferation.

Alternatively viewed, the present invention provides a short activatingRNA which up-regulates albumin production and thereby inhibits cellproliferation for use in therapy, preferably for use in treating orpreventing a hyperproliferative disorder.

Also provided is an in vitro, ex vivo or in vivo method of inhibitingcell proliferation, comprising contacting a cell (sample), tissue(sample), organ or subject with a short activating RNA whichup-regulates albumin production and thereby inhibits cell proliferation.

Also provided is a method of treating a subject having a conditioncharacterised by hypoalbuminemia, comprising administering to saidsubject a short activating RNA which up-regulates albumin production.

Alternatively viewed, the present invention provides a short activatingRNA which up-regulates albumin production for use in therapy, preferablyfor use in treating a disorder characterised by hypoalbuminemia.

Also provided is an in vitro, ex vivo or in vivo method of up-regulatingalbumin production by a cell, comprising contacting a cell (sample),tissue (sample), organ or subject with a short activating RNA whichup-regulates albumin production.

In another aspect, the invention provides an saRNA capable ofupregulating albumin production. Optional features of the saRNAmolecules are described elsewhere herein.

The saRNAs of the invention may advantageously exert a dual effect,namely inhibit cell hyperproliferation and increase albumin levels. Whenadministered to a subject suffering from a hyperproliferative disorder,the saRNAs not only reduce proliferation of the hyperproliferativecells, but they also increase albumin levels which may have a variety ofpositive outcomes such as improving liver function. When administered toa subject suffering from hypoalbuminemia, the saRNAs increase albuminlevels and thereby alleviate the hypoalbuminemia, and they also help toprevent the development of a hyperproliferative disorder. A number ofliver disorders, particularly cirrhosis, are characterised byhypoalbuminemia and particularly if left untreated typically increasethe risk of the subject developing cancer. The present inventionprovides particularly advantageous treatments for such liver disorders,the treatments alleviating the hypoalbuminemia and reducing the risk ofcancer. Thus, provided are saRNAs which simultaneously treat conditionscharacterised by hypoalbuminemia and hyperproliferative disorders.

“Capable of upregulating albumin production” means that the saRNAupregulates albumin production when inside a cell that has the necessarymachinery for producing albumin and preferably naturally produces somealbumin. Advantageously, upregulating albumin production inhibits cellproliferation, particularly hyperproliferation, so said saRNA is capableof inhibiting cell proliferation, particularly hyperproliferation.“Capable of inhibiting cell proliferation” means that the saRNA inhibitscell proliferation when inside a cell that has the necessary machineryfor cell proliferation.

In another aspect, the invention provides an ex vivo or in vitro cellcomprising an saRNA molecule of the invention. In another aspect, theinvention provides an ex vivo or in vitro cell in which albuminproduction has been upregulated by a method disclosed herein. Thus, sucha cell is obtained or obtainable by the methods disclosed herein. Alsoprovided is such a cell for use in therapy, preferably the treatment ofa condition characterised by hypoalbuminemia, or any other type of liverdisease.

A “hyperproliferative cell” may be any cell that is proliferating at arate that is abnormally high in comparison to the proliferating rate ofan equivalent healthy cell (which may be referred to as a “control”). An“equivalent healthy” cell is the normal, healthy counterpart of a cell.Thus, it is a cell of the same type, e.g. from the same organ, whichperforms the same functions(s) as the comparator cell. For example,proliferation of a hyperproliferative hepatocyte should be assessed byreference to a healthy hepatocyte, whereas proliferation of ahyperproliferative prostate cell should be assessed by reference to ahealthy prostate cell.

By an “abnormally high” rate of proliferation, it is meant that the rateof proliferation of the hyperproliferative cells is increased by atleast 20, 30, 40%, or at least 45, 50, 55, 60, 65, 70, 75%, or at least80%, as compared to the proliferative rate of equivalent, healthy(non-hyperproliferative) cells. The “abnormally high” rate ofproliferation may also refer to a rate that is increased by a factor ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, or by a factor of at least 15, 20,25, 30, 35, 40, 45, 50, or by a factor of at least 60, 70, 80, 90, 100,compared to the proliferative rate of equivalent, healthy cells.

Examples of hyperproliferative cells include cancerous cells (includingcarcinomas, sarcomas, lymphomas and blastomas). Such cancerous cells maybe benign or malignant. Hyperproliferative cells may also result from anautoimmune condition such as rheumatoid arthritis, inflammatory boweldisease, or psoriasis. Hyperproliferative cells may also result withinpatients with an oversensitive immune system coming into contact with anallergen. Such conditions involving an oversensitive immune systeminclude, but are not limited to, asthma, allergic rhinitis, eczema, andallergic reactions, such as allergic anaphylaxis.

The term “hyperproliferative cell” as used herein does not refer to acell which naturally proliferates at a higher rate as compared to mostcells, but is a healthy cell. Examples of cells that are known to divideconstantly throughout life are skin cells, cells of the gastrointestinaltract, blood cells and bone marrow cells. However, when such cellsproliferate at a higher rate than their healthy counterparts, then theyare hyperproliferative.

A “hyperproliferative disorder” may be any disorder which involveshyperproliferative cells as defined above. Examples ofhyperproliferative disorders include neoplastic disorders such ascancer, psoriatic arthritis, rheumatoid arthritis, gastrichyperproliferative disorders such as inflammatory bowel disease, skindisorders including psoriasis, Reiter's syndrome, pityriasis rubrapilaris, and hyperproliferative variants of the disorders ofkeratinization.

Cancer represents a hyperproliferative disorder of particular interest,and all types of cancers, including e.g. solid tumours andhaematological cancers are included. Representative types of cancerinclude cervical cancer, uterine cancer, ovarian cancer, kidney cancer,gallbladder cancer, liver cancer, head and neck cancer, squamous cellcarcinoma, gastrointestinal cancer, breast cancer, prostate cancer,testicular cancer, lung cancer, non-small cell lung cancer,non-Hodgkin's lymphoma, multiple myeloma, leukemia (such as acutelymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenousleukemia, and chronic myelogenous leukemia), brain cancer (e.g.astrocytoma, glioblastoma, medulloblastoma), neuroblastoma, sarcomas,colon cancer, rectum cancer, stomach cancer, anal cancer, bladdercancer, endometrial cancer, plasmacytoma, lymphomas, retinoblastoma,Wilm's tumor, Ewing sarcoma, melanoma and other skin cancers. Livercancer or prostrate cancer are preferred. The liver cancer may includeor consist of cholangiocarcinoma, hepatoblastoma, haemangiosarcoma, orhepatocellular carcinoma (HCC). HCC is of particular interest.

Preferably, tumour development and/or growth is inhibited. In apreferred embodiment, solid tumours are treated. In another preferredembodiment, metastasis is prevented.

By the “inhibition of cell proliferation” or “reduced proliferation” ismeant that proliferation is reduced or stops altogether. Thus, “reducingproliferation” is an embodiment of “inhibiting proliferation”.Proliferation of a cell is reduced by at least 20%, 30% or 40%, orpreferably at least 45, 50, 55, 60, 65, 70 or 75%, even more preferablyat least 80, 90 or 95% in the presence of the oligonucleotides of theinvention compared to the proliferation of said cell prior to treatmentwith the oligonucleotides of the invention, or compared to theproliferation of an equivalent untreated cell. In embodiments whereincell proliferation is inhibited in hyperproliferative cells, the“equivalent” cell is also a hyperproliferative cell. In preferredembodiments, proliferation is reduced to a rate comparable to theproliferative rate of the equivalent healthy (non-hyperproliferative)cell. Alternatively viewed, a preferred embodiment of “inhibiting cellproliferation” is the inhibition of hyperproliferation, or modulatingcell proliferation to reach a normal, healthy level of proliferation.

The skilled person is fully aware of how to identify ahyperproliferative cell. The presence of hyperproliferative cells withinan animal may be identifiable using scans such as X-rays, MRI or CTscans. The hyperproliferative cell may also be identified, or theproliferation of cells may be assayed, through the culturing of a samplein vitro using cell proliferation assays, such as MTT, XTT, MTS or WST-1assays. Cell proliferation in vitro can also be determined using flowcytometry.

The cell proliferation assays listed above all work by the sameprinciple; a stable tetrazolium salt is cleaved to form a solubleformazan by a complex cellular mechanism that occurs primarily at thecell surface. This bioreduction is largely dependent on the glycolyticproduction of NAD(P)H in viable cells. Therefore, the amount of formazandye formed directly correlates to the number of metabolically activecells in the culture. Cells, grown in a tissue culture plate, areincubated with the reagent for approximately 0.5-4 hours. After thisincubation period, the formazan dye formed is quantitated with ascanning multi-well spectrophotometer (ELISA reader). The measuredabsorbance directly correlates to the number of viable cells.

Tumor development or growth may be assayed using one or more knowntechniques, such as measurement of tumor size using an external caliper,calculation of tumor volume e.g. by use of the ellipsoid formula,computed tomography (CT), micro CT, positron emission tomography (PET),microPET, magnetic resonance imaging (MRI), immunohistochemistry and/oroptical imaging such as bioluminescence imaging (BLI) or fluorescenceimaging (FLI). In animal studies, once the animal has been sacrificedtumour volume may also be determined by measuring water displacementvolume and tumour weight may be determined using scales.

The method of the present invention preferably reduces tumour volume,preferably by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%.Preferably, the development of one or more new tumours is inhibited,e.g. a subject treated according to the invention develops fewer and/orsmaller tumours. By “fewer tumours” is meant that he develops a smallernumber of tumours than an equivalent subject over a set period of time.Preferably, he develops at least 1, 2, 3, 4 or 5 fewer tumours than anequivalent control (untreated) subject. By “smaller” tumours is meantthat the tumours are at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%smaller in weight and/or volume than tumours of an equivalent subject.

The set period of time may be any suitable period, e.g. 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 months or years.

Preferably, the onset of cancer is prevented or delayed. This may beassessed by reference to an equivalent control (untreated) subject.

An “equivalent subject” may be e.g. a subject of similar age, sex andhealth such as liver health or cancer stage, or the same subject priorto treatment according to the invention. The equivalent subject is“untreated” in that he does not receive treatment with an saRNAaccording to the invention. However, he may receive a conventionalanti-cancer treatment, provided that the subject who is treated with thesaRNA of the invention receives the same or equivalent conventionalanti-cancer treatment.

Albumin expression may be assayed through the use of well-establishedtechniques that the skilled person would be well aware of Theup-regulation of the messenger RNA that encodes albumin may bedetermined using reverse transcriptase polymerase chain reaction(RT-PCR), preferably semi-quantitative or quantitative PCR (qRT-PCR).This method involves reverse transcribing the isolated mRNA into cDNAbefore carrying out real-time amplification cycles in the presence of ahybridization reporter probe for quantitative analysis. Albumin mRNAexpression can also be determined using Northern blot analysis.

An up-regulation of the production of the albumin protein can bedetermined using an enzyme-linked immunosorbent assay (ELISA). ThisELISA may be a sandwich ELISA, such as the AssayMax Albumin ELISA kit.Here, the sample is applied to a microplate that has been pre-coatedwith an antibody specific to albumin. After washing, which removes anynon-albumin protein, the remaining albumin is sandwiched by usinganother specific antibody. Excess antibody is washed before a secondaryantibody is applied which has a detectable label. Protein expression canalso be determined using Western blot analysis or protein massspectrometry.

By “up-regulating albumin expression”, it is meant that the expressionof the albumin protein is increased by at least 20, 30, 40%, morepreferably at least 45, 50, 55, 60, 65, 70, 75%, even more preferably atleast 80% in the presence of the saRNA oligonucleotides of the inventioncompared to the expression of albumin in the absence of the saRNAoligonucleotide. In a further preferable embodiment, the expression ofthe albumin protein is increased by a factor of at least 2, 3, 4, 5, 6,7, 8, 9, 10, more preferably by a factor of at least 15, 20, 25, 30, 35,40, 45, 50, even more preferably by a factor of at least 60, 70, 80, 90,100, in the presence of the saRNA oligonucleotides of the inventioncompared to the expression of albumin in the absence of the saRNAoligonucleotide. In another preferred embodiment, the up-regulationwould relate to both the synthesis and the processing of the serumalbumin protein into its active form.

The upregulation of albumin production may be assayed by reference toalbumin expression by the test cell prior to treatment with the saRNAsof the invention, or by reference to albumin expression by an equivalentuntreated cell (control).

Preferably, the methods of the invention also result in secretion ofalbumin, more preferably in increased secretion of albumin. Preferably,intracellular albumin levels and/or serum levels of albumin areincreased.

In some embodiments, the methods of the invention result in upregulationof the expression of CEBPA and/or HNF4A, but in other embodiments, themethods of the invention result in downregulation of CEBPA and/or HNF4A.

In some embodiments, the methods of the invention result indownregulation of a fetoprotein and/or hepatocyte growth factor.

In some embodiments, the methods disclosed herein may include a step ofassaying cell proliferation and/or assaying albumin production beforeand/or after administration of the saRNA to a subject. For example, themethods may include a step of determining that albumin expression hasincreased and/or that proliferation of hyperproliferative cells, e.g.cancer cells, has decreased as a result of a treatment according to thepresent invention.

Hypoalbuminemia is a condition where albumin within blood serum isinsufficient or abnormally low. There are many conditions that arecharacterised by hypoalbuminemia, including conditions which causehypoalbuminemia and conditions which are the cause of hypoalbuminemia.Such conditions include, but are not limited to, liver diseases, such ashepatitis B and C, viral infections of the liver, alcoholic liverdisease such as fatty liver, liver cirrhosis or alcoholic hepatitis,conditions that cause an excessive level of albumin to be excreted, suchas nephrotic syndrome, conditions that cause albumin to be lost throughthe bowels, such as Menetrier's disease, burns patients, who losealbumin through plasma loss, and conditions that cause a redistributionof albumin, such as oedema. Such conditions may benefit from increasedlevels of albumin, which may be provided by the saRNA oligonucleotidesof the invention.

An insufficient level of albumin is defined by a level of albumin thatfalls outside the levels considered healthy for that animal. Forexample, in humans, an insufficient level of albumin would be consideredas a level below 38 g/L or 35 g/L, or even below 32 or 30 g/L.

Liver disease or poor liver function is thus typically characterised byhypoalbuminemia, so it may be useful to assess liver function todetermine whether a subject may benefit from a therapy provided by thepresent invention. Liver function assessments may also provide a usefulway of determining the success of a particular treatment.

Methods that may be used to diagnose a diseased liver that may benefitfrom the treatments of the invention, or to determine the success of aparticular treatment, are well known in the art. Liver function testsmay be carried out through analysing a blood sample obtained from ananimal. Proteins that would be quantified in such liver function testsmay include bilirubin, aspartate aminotransferase (AST), alanineaminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyltranspeptidase (GGT), total protein and globulin protein, as well asserum albumin. These proteins may be referred to as “liver functionmarkers” or “liver health markers”. Below is a Table that shows thehealthy ranges of the above proteins in humans.

TABLE 3 Minimum Maximum Protein concentration concentration Bilirubin 0μmol/L 20 μmol/L AST 0 U/L 45 U/L ALT 0 U/L 45 U/L ALP 30 U/L 120 U/LGGT 0 U/L 45 U/L Total Protein 60 g/L 80 g/L Globulin Protein 20 g/L 32g/L Serum Albumin 38 g/L 55 g/L

Many of the above proteins are not liver-specific indicators of disease.However, elevated GGT alone usually indicates some form of liverdisease. When some of the protein levels are analysed in combination,they can be an accurate means of determining specific liver diseases.For example, an elevated GGT, a decrease in ALT and a decrease in ALP incombination could be an indication of alcoholic liver disease or fattyliver disease. An elevated GGT, an elevated ALT and a decrease in ALP incombination could be an indication of hepatitis, but can also be anindication of fatty liver disease.

In some embodiments, the methods of the invention include a step ofdiagnosing a liver disease/diagnosing that a subject may benefit from ansaRNA or cell of the invention. Said step may comprise carrying out oneor more of these liver function tests. Alternatively or in addition, themethods may include a step of analysing liver function afteradministration of the saRNA or cell of the invention. Said step maycomprise carrying out one or more of these liver function tests.Preferably, after said treatment an improvement in at least one of saidliver function markers is determined.

For example, at least 1 of bilirubin, AST, ALT, ALP and GGT may show adecrease that is 1, 2, 3, 4 or 5 fold, or an increase by at least 10,20, 30, 40, 50, 60, 70, 80 or 90%. Preferably, at least 2, 3, 4 or allof these markers show such a decrease. Thus, the methods of theinvention preferably result in a blood bilirubin concentration of lessthat 20, 15, 10 or 5 μmol/L; a blood AST concentration of less than 45,40, 30, 25, 20, 15, 10 or 5 U/L; a blood ALT concentration of less than45, 40, 30, 25, 20, 15, 10 or 5 U/L; a blood GGT concentration of lessthan 45, 40, 30, 25, 20, 15, 10 or 5 U/L; and/or a blood ALPconcentration of less than 120, 100, 80, 70, 60, 50 or 40 U/L, butpreferably no less than 30 U/L.

The methods of the invention preferably result in a blood albuminconcentration of at least 32, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 g/L, e.g. 38-55, 40-55, 42-55, 45-55 g/L.

Effective treatments according to the present invention may mean thatthe disease that is being treated, be that one involvinghyperproliferative cells or hypoalbuminemia, is cured. However, aneffective “treatment” encompasses any outcome which is of benefit to thepatient, be it temporary or permanent. The treatment may affect theunderlying condition, and/or it may affect the symptoms. Thus, purelysymptomatic relief is also contemplated.

Prevention according to the present invention means that theprophylactic administration of the saRNA oligonucleotides or cells ofthe present invention prevents, reduces the risk of, or delays the onsetof the development a condition characterised by hyperproliferative cellsor an insufficient level of albumin. Such prophylactic administrationmay be used for patients that are at risk of developing a conditioncharacterised by hyperproliferative cells or hypoalbuminemia.Prophylactic administration may be effective over the lifetime of thepatient. However, prophylactic administration may also need to berepeated over the lifetime of the patient, or until the condition thatputs the patient at risk can be alleviated.

In the methods of the invention, cells are contacted with a short RNAmolecule of the present invention. The cells may be in vitro, e.g. anestablished cell line or a sample having previously been obtained from asubject, or they may be in vivo in a subject. The short RNA moleculescan be administered to cells in vitro or in vivo by using any suitabledelivery reagents in conjunction with the present short RNAs. Suchsuitable delivery reagents include the Mirus Transit TKO lipophilicreagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), virus-based particles, electroporation dendrimers orliposomes. A preferred delivery reagent is a liposome. A variety ofmethods are known for preparing liposomes.

Another preferred deliver reagent is a dendrimer. Dendrimers aredescribed in Singha et al. (2011), Nucleic Acid Ther. 21:133, the entiredisclosure of which is herein incorporated by reference. Dendrimers areregular and highly branched macromolecules. Preferably, the dendrimer isa polycationic dendrimer, such as PAMAM dendrimers. Without wishing tobe bound by theory, PAMAM dendrimers bear primary amine groups on theirsurface which facilitate nucleic acid binding, and contain tertiaryamino groups which enhance the release of the nucleic acid in thecytoplasm.

The step of contacting cells with saRNAs may also be referred to as“transfection”.

Particularly preferably, the liposomes encapsulating the present shortRNAs are modified so as to avoid clearance by the mononuclear macrophageand reticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

Recombinant plasmids which can express the short RNAs can also beadministered directly or in conjunction with a suitable deliveryreagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin;lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.Recombinant viral vectors which express the short RNA and methods fordelivering such vectors to a cell are known within the art.

Preferably said contacting step is performed more than once, preferablyevery 3 days, although it may also be daily, or every 2, 4 or 5 days.The contacting is preferably performed for at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15 days, about 6 or 9 days being preferred.Contacting at regular intervals over a period of weeks or months is alsocontemplated.

In the methods disclosed herein, if more than one target gene isup-regulated then the short RNAs used to up-regulate the differenttarget genes may be administered at different frequencies and fordifferent lengths of time. The particular administration regimens to beused can be readily determined by one of ordinary skill in the art tosuit his desired purpose, particular starting cell type and deliverymethod. By way of example, picoMolar concentrations of the short RNAmolecules of the present may be used.

The short RNA of the invention may be provided alone or in combinationwith other active agent(s) known to have an effect in the particularmethod being considered. The other active agent(s) may be administeredsimultaneously, separately or sequentially with the short RNA of theinvention. Thus, it is possible to use a single short RNA of theinvention, a combination of two or more short RNAs of the invention or acombination of said short RNA(s) and other active substance(s).

The methods of the invention are preferably carried out on cells orsubjects that are animal, more preferably mammalian, e.g. mouse, rat,monkey, dog, cow, sheep, most preferably human, although optionally thecell or subject is non-human. The cell or subject may be embryonic, butis preferably adult.

It must be appreciated that the methods of the inventions may not, andindeed need not, achieve the upregulation of albumin production in allof the cells within a population of cells that is contacted with ansaRNA of the invention. Thus, out of a population of cells subjected tothe method of the present invention, i.e. contacted with an saRNA of theinvention, preferably at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%,may be induced to produce more albumin, although in some embodimentsalbumin production is up-regulated in at least 95 or 99% of the cells.

In vitro and in vivo methods may be carried out using (i) a cell whichnaturally produces albumin, such as a hepatocyte; (ii) a cell which hasthe potential to differentiate into a cell which naturally producesalbumin, such as a stem cell; or (iii) a cell which does not normallyproduce albumin, such a somatic non-liver cell. Hepatocytes or stemcells are preferred. The stem cell may be totipotent, pluripotent ormultipotent, e.g. Haemopoietic stem cells (HSC), mesenchymal stem cells(MSC) or induced pluripotent stem cells.

WO2005/059113 discloses a particularly advantageous type of pluripotentstem cell. This stem cell can be directly isolated from bone marrowand/or blood, e.g. peripheral blood, or from material taken from theumbilical cord or placenta, and has the unique ability to differentiateinto ectodermal, mesodermal and endodermal cells. These cells are thusclearly multipotent or pluripotent, if not totipotent. Therefore, thestem cells described in WO2005/059113 provide a useful source of cellsfor tissue transplantation that may be used in an autologous(self-to-self) manner.

The cells disclosed in WO2005/059113 are known in the art as“OmniCytes”. The teachings of WO2005/059113 are incorporated herein intheir entirety by reference. OmniCytes are stem cells which are CD34+,capable of self regeneration and capable of differentiation intoectodermal, mesodermal and endodermal cells, including haemopoieticcells. As mentioned above, they can be directly isolated from bonemarrow and/or blood. They are further characterised by their ability toadhere to plastic (e.g. the plastic of standard tissue culture vessels)during culturing. Suitable vessels are those manufactured by CorningIncorporated, New York, USA.

OmniCytes may be further characterised by the fact that they do notrequire feeder layers, i.e. cells (typically inactivated by gammairradiation which supply important metabolites without further growth ordivision of their own) which support the growth of the stem cells.

OmniCytes can be further characterised as obtainable by:

-   a) enrichment of a tissue or blood sample for CD34⁺ cells;-   b) contacting the sample with a solid support and harvesting the    cells which adhere to said solid support.

Suitable tissue or blood samples include, bone marrow, peripheral blood,umbilical cord blood or tissue, placenta and samples obtained fromliposuction.

More particularly, they are obtainable by:

-   subjecting a tissue or blood sample (preferably haemopoietic tissue    such as blood or a bone marrow sample) to density gradient    separation;-   exposing low density cells to an affinity ligand for CD34    (preferably attached to paramagnetic beads);-   recovering cells attached to said CD34 ligand;-   exposing the CD34+ subpopulation to tissue culture grade plastic;    and-   recovering CD34+ cells adherent to the plastic.

Omnicytes are preferably adult, so non-foetal.

A sample of OmniCytes was deposited with ECACC at Porton Down,Salisbury, SP4 0JG on 24 Sep. 2004 under accession number 04092401. Thedeposit was made by Professor Myrtle Gordon of Willow Tree Cottage,Spinning Wheel Lane, Binfield, Berkshire RG42 5QH, Great Britain and thecell line was given the name “Stem Cell OmniCyte”. On 14 Jun. 2012Professor Myrtle Gordon authorised Mina Therapeutics Limited to refer tothe deposited biological material in any and all Patent Applications,and she gave her unreserved and irrevocable consent to the depositedmaterial being made available to the public in accordance with Rule 33EPC.

As set out in the Examples, the methods of the invention allow thegeneration of cells which produce albumin. More specifically, theyproduce more albumin than their untreated counterparts, so theyoverproduce albumin, or they are cells that do not typically producealbumin but which have been induced to produce albumin. Thus, in afurther aspect there is provided a cell which produces or overproducesalbumin. The cell is obtainable by the methods disclosed herein.Preferably, the cell is obtained by inducing a CD34+ stem cell such asan OmniCyte to produce albumin.

The cell is preferably ex vivo, i.e. not part of a living organism.Optionally, the cell may be referred to as “in vitro” or “isolated”.

Uses of such cells in therapy represents a further aspect of theinvention.

Optionally, the cell of the present invention and saRNA of the presentinvention may be used in combination in the therapeutic applicationsdisclosed herein. “In combination” includes separate, simultaneous orsequential administration.

Alternatively viewed, the present invention provides a method oftreatment comprising administering to a subject in need thereof atherapeutically effective amount of an albumin-producing cell and/or atherapeutically effective amount of an saRNA as defined herein.

The saRNAs may be designed to be complementary to a target transcript,and they may exert their effect on albumin production by down-regulatinga target antisense RNA transcript.

The target RNA transcript is preferably transcribed from a locus up to100, 80, 60, 40, 20 or 10 kb upstream of the target gene's transcriptionstart site, or from a locus up to 100, 80, 60, 40, 20 or 10 kbdownstream of the target gene's transcription stop site. Optionally, theRNA transcripts are transcribed from a locus up to 1 kb upstream of thetarget gene's transcription start site or from a locus up to 1 kbdownstream of the target gene's transcription stop site. Optionally, theRNA transcripts are transcribed from a locus up to 500, 250 or 100nucleotides upstream of the target gene's transcription start site orfrom a locus up to 500, 250 or 100 nucleotides downstream of the targetgene's transcription stop site, more preferably the locus is no morethan 500 nucleotides upstream or downstream from the target gene'stranscription start site. Most preferably the target RNA transcript istranscribed from a locus up to 500 nucleotides upstream or up to 500nucleotides downstream of the target gene's transcription start site.

The term “is transcribed from [a particular locus]” in the context ofthe target RNA transcripts of the invention means “the transcriptionstart site of the target RNA transcript is found [at the particularlocus]”. Preferably both of the transcription start site and thetranscription stop site of the target RNA transcript are, separately,located either up to 100 kb upstream of the target gene's transcriptionstart site or up to 100 kb downstream of the target gene's transcriptionstop site. The preferred embodiments described above in relation to thelocation from which the target RNA transcript is transcribed applymutatis mutandis to the location of the target RNA transcript'stranscription stop site.

The target RNA transcript is complementary to the coding strand of thegenomic sequence, and any reference herein to “genomic sequence” isshorthand for “coding strand of the genomic sequence”.

Thus, the target RNA transcript comprises a sequence which is antisenseto a genomic sequence located between 100, 80, 60, 40, 20 or 10 kbupstream of the target gene's transcription start site and 100, 80, 60,40, 20 or 10 kb downstream of the target gene's transcription stop site.More preferably, the target RNA transcript comprises a sequence which isantisense to a genomic sequence located between 1 kb upstream of thetarget gene's transcription start site and 1 kb downstream of the targetgene's transcription stop site. More preferably, the target RNAtranscript comprises a sequence which is antisense to a genomic sequencelocated between 500, 250 or 100 nucleotides upstream of the targetgene's transcription start site and ending 500, 250 or 100 nucleotidesdownstream of the target gene's transcription stop site. Optionally thetarget RNA transcript comprises a sequence which is antisense to agenomic sequence which includes the coding region of the target gene.Most preferably, the target transcript comprises or consists of asequence which is antisense to a genomic sequence that overlaps with thetarget gene's promoter region. Thus, the target RNA transcriptpreferably comprises a sequence which is antisense to the promoterregion.

Genes may possess a plurality of promoter regions, in which case thetarget RNA transcript may overlap with one, two or more of the promoterregions. Online database of annotated gene loci may be used to identifythe promoter regions of genes.

For any given promoter region, the entire promoter region does not haveto be overlapped, it is sufficient for a subsequence within the promoterregion to be overlapped by the target RNA transcript, i.e. the overlapcan be a partial overlap. Similarly, the entire target RNA transcriptneed not be antisense to the sequence within the promoter region, it isonly necessary for the target RNA transcript to comprise a sequencewhich is antisense to the promoter region.

The region of overlap between the target RNA transcript and the promoterregion of the target gene may be as short as a single nucleotide inlength, although it is preferably at least 15 nucleotides in length,more preferably at least 25 nucleotides in length, more preferably atleast 50 nucleotides in length, more preferably at least 75 nucleotidesin length, most preferably at least 100 nucleotides in length. Each ofthe following specific arrangements are intended to fall within thescope of the term “overlap”:

a) The target RNA transcript and the target gene's promoter region areidentical in length and they overlap (i.e. they are complementary) overtheir entire lengths.

b) The target RNA transcript is shorter than the target gene's promoterregion and overlaps over its entire length with the target gene'spromoter region (i.e. it is complementary over its entire length to asequence within the target gene's promoter region).

c) The target RNA transcript is longer than the target gene's promoterregion and the target gene's promoter region is overlapped fully by it,i.e. the target gene's promoter region is complementary over its entirelength to a sequence within the target RNA transcript).

d) The target RNA transcript and the target gene's promoter region areof the same or different lengths and the region of overlap is shorterthan both the length of the target RNA transcript and the length of thetarget gene's promoter region.

The above definition of “overlap” applies mutatis mutandis to thedescription of other overlapping sequences throughout the description.Clearly, if an antisense RNA transcript is described as overlapping witha region of the target gene other than the promoter region then thesequence of the transcript is complementary to a sequence within thatregion rather than within the promoter region.

Preferably the RNA transcript comprises a sequence which is antisense toa genomic sequence which comprises the target gene's transcription startsite. In other words, preferably the target RNA transcript comprises asequence which overlaps with the target gene's transcription start site.

The target RNA transcript is preferably at least 1 kb, more preferablyat least 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g. 20, 25, 30, 35 or 40 kblong.

The term “sense” when used to describe a nucleic acid sequence in thecontext of the present invention means that the sequence has identity toa sequence on the coding strand of the target gene. The term “antisense”when used to describe a nucleic acid sequence in the context of thepresent invention means that the sequence is complementary to a sequenceon the coding strand of the target gene.

The “coding strand” of a gene is the strand which contains the codingsequence for the gene's mRNA. The “template strand” of a gene is thestrand which does not contain the coding sequence for the gene's mRNA.

Preferably the target RNA transcript comprises a sequence which is atleast 75%, preferably at least 85%, more preferably at least 90%, stillmore preferably at least 95% complementary along its full length to asequence on the coding strand of the target gene.

The present invention provides saRNA molecules which can effectively andspecifically down-regulate such target transcripts. This can be achievedby the saRNA molecules having a high degree of complementarity to asequence within the target RNA transcript. The short RNA will have nomore than 5, preferably no more than 4 or 3, more preferably no morethan 2, still more preferably no more than 1, most preferably nomismatches with a region of a target RNA transcript.

Preferably, the saRNA comprises a sequence of at least 13 nucleotideswhich has at least 95, 98, 99 or 100% complementarity to a region of thetarget transcript. Preferably, said sequence which has at least 95, 98,99 or 100% complementarity to a region of the target transcript is atleast 15-20, e.g. at least 15, 16, 17,18 or 19 nucleotides in length,preferably 18-22 or 19 to 21, most preferably exactly 19. As mentionedelsewhere, the saRNA may also comprise a 3′ tail.

In one embodiment, the short RNA molecules of the present invention mayhave siRNA-like complementarity to a region of the target transcript;that is, 100% complementarity between nucleotides 2-6 from the 5′ end ofthe guide strand in the saRNA duplex and a region of the targettranscript. Other nucleotides of the short RNA molecule may, inaddition, have at least 70, 80, 90, 95, 99 or 100% complementarity to aregion of the target transcript. For example, nucleotides 7 (countedfrom the 5′ end) until the 3′ end may have least 70, 80, 90, 95, 99 or100% complementarity to a region of the target transcript.

The sequence within the target RNA transcript to which the saRNAmolecules of the present invention preferably have a high degree ofcomplementarity is preferably antisense to a genomic sequence that is upto 500 nucleotides upstream or downstream of the target gene'stranscription start site. Preferably, it overlaps the target gene'spromoter.

The skilled person will appreciate that it is convenient to define thesaRNA by reference to the target transcript, regardless of the mechanismby which the saRNA upregulates albumin production. However, the saRNAmay alternatively be defined by reference to the target gene. The targettranscript is complementary to a genomic region on the coding strand ofthe target gene, and the saRNA is in turn complementary to a region ofthe target transcript, so the saRNA may be defined as having sequenceidentity to a region on the coding strand of the target gene. All of thefeatures discussed herein with respect to the definition of the saRNA byreference to the target transcript apply mutatis mutandis to thedefinition of the saRNA by reference to the target gene so anydiscussion of “complementarity” to the “target transcript” should beunderstood to include “identity” to the “genomic sequence”. Thus, thesaRNA preferably has a high percent identity, e.g. at least 75, 80, 85,90, 95, 98 or 99, preferably 100% identity, to a genomic sequencesurrounding the target gene's transcription start site. The distancesthat the genomic sequence may have from the TSS are discussed above. Itis preferable that the genomic sequence is up to 500 nucleotidesupstream or downsteam of the TSS. Most preferably, it overlaps thetarget gene's promoter. Thus, the saRNA preferably has sequence identityto a sequence that overlaps the promoter region of the target gene.

Preferably the “short” RNA molecule of the invention is from 13nucleotides to 30 nucleotides in length, preferably from 15 or 17 to 30nucleotides, more preferably 16 to 25 nucleotides in length, still morepreferably 17 to 21 nucleotides in length, most preferably 19, 20, 21 or22 nucleotides in length. In other words, the short RNA molecules maycomprise a first strand of a length discussed above. If a 3′ tail ispresent, the strand may be longer, preferably 19 nucleotides plus a 3′tail, which is preferably UU or UUU.

The short RNA molecule may be single or, preferably, double stranded.Double stranded molecules comprise a first strand and a second strand.If double stranded, preferably each strand of the duplex is at least 14,more preferably at least 18, e.g. 19, 20, 21 or 22 nucleotides inlength. Preferably the duplex is hybridised over a length of at least12, more preferably at least 15, more preferably 17, still morepreferably at least 19 nucleotides. Each strand may be exactly 19nucleotides in length. If a 3′ tail is present, the strand may belonger, preferably 19 nucleotides plus a 3′ tail, which is preferably UUor UUU.

Preferably the duplex length is less than 30 nucleotides since duplexesexceeding this length may have an increased risk of inducing theinterferon response. The strands forming the dsRNA duplex may be ofequal or unequal lengths.

Most preferably the short RNA molecule is a short interfering RNA(siRNA) molecule.

Optionally the short RNA molecules are dsRNA molecules which consist ofthe two strands stably base-paired together with a number of unpairednucleotides at the 3′ end of each strand forming 3′ overhangs. Thenumber of unpaired nucleotides forming the 3′ overhang of each strand ispreferably in the range of 1 to 5 nucleotides, more preferably 1 to 3nucleotides and most preferably 2 nucleotides. The 3′ overhang may beformed of the 3′ tail mentioned above, so the 3′ tail may be the 3′overhang.

All references to sequence complementarity or identity used herein referto the whole length of the short RNA molecule unless specifically statedotherwise.

The short RNA may include a very short 3′ or 5′ sequence which is notcomplementary to the target RNA transcript. Preferably, such a sequenceis 3′. Said sequence may be 1-5 nucleotides in length, preferably 2-3,e.g. 2 or 3. Said sequence preferably comprises or consists of uracil,so most preferably it is a 3′ stretch of 2 or 3 uracils. Thisnon-complementary sequence may be referred to as “tail”. Thus, the shortRNA preferably consists of (i) a sequence having at least 95%complementarity to a region of the target RNA; and (ii) a 3′ tail of 1-5nucleotides, which preferably comprises or consists of uracil residues.The short RNA will thus typically have complementarity to a region ofthe target RNA transcript over its whole length, except for the 3′ tail,if present. As mentioned above, instead of “complementary to the targetRNA transcript” the saRNA may be defined as having “identity” to thecoding strand of the relevant genomic sequence.

Preferred sequences of suitable saRNAs of the invention are provided inTable 1. Thus, provided are short RNAs having a first strand comprisingor consisting of a sequence selected from SEQ ID NOs: 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. Optionally, the short RNAmay comprise a 3′ tail at the 3′ end of any of these sequences.

Single stranded short RNA molecules only consist of a first strand,whereas double stranded short RNA molecules also have a second strand.The short RNAs may thus have a second strand comprising or consisting ofa sequence selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33 and 35.

A short RNA having a first strand of SEQ ID NO:14 and a second strand ofSEQ ID NO: 13 was shown to be particularly effective in treating cancer,so this is particularly preferred.

Table 1 indicates preferred pairings, each row representing a preferredpairing. Thus, for example, the short RNA preferably has a first strandcomprising or consisting of a sequence of SEQ ID NO: 14, optionally witha 3′ tail, and a second strand or consisting of a sequence of SEQ ID NO:13 optionally with a 3′ tail.

Any of the short RNA sequences disclosed herein may optionally includesuch a 3′ tail. Thus, any of the sequences disclosed in the Tables mayoptionally include such a 3′ tail.

As used herein, the term “RNA” means a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′ position of a beta-D-ribo-furanose moiety. Theterms include double stranded RNA, single stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the present invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

The term “double stranded RNA” or “dsRNA” as used herein refers to aribonucleic acid duplex.

The term “short interfering RNA” or “siRNA” as used herein refers to anucleic acid molecule capable of modulating gene expression through RNAivia sequence-specific-mediated cleavage of one or more target RNAtranscripts. Typically, in RNAi the RNA transcript is mRNA and socleavage of this target results in the down-regulation of geneexpression. In this invention however, up-regulation or down-regulationof the target gene can be achieved by cleavage of RNA transcripts whichare antisense or sense to the target gene of interest respectively.

By “complementarity” and “complementary” are meant that a first nucleicacid can form hydrogen bond(s) with a second nucleic acid for example byWatson-Crick base pairing. A nucleic acid which can form hydrogenbond(s) with another nucleic acid through non-Watson-Crick base pairingalso falls within the definition of having complementarity. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary).

By “identity”, “identical” or “sequence identity” is meant that a firstnucleic acid is identical in sequence to a second nucleic acid sequence.A percent identity indicates the percentage of residues in a firstnucleic acid molecule that are identical to a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%,90%, and 100% identical).

Sequence alignments and percent identity or percent complementaritycalculations may be determined using any method or tool known in the artincluding but not limited to the Megalign program of the LASARGENEbioinformatics computing suite (DNASTAR Inc., Madison, W1), the ClustalV method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) andthe BLAST 2.0 suite of programs. Software for performing BLAST analysesis publicly available, e.g., through the National Center forBiotechnology Information. The skilled man will be able to set theparameters of these tools to suit his desired purpose.

When assessing the identity or complementarity of a first and secondnucleic acid sequence wherein one sequence is a DNA sequence and theother is an RNA sequence, it must be borne in mind that RNA sequencescomprise uracil whereas DNA sequences would comprise thymine instead.Therefore, in these instances when assessing sequence identity, a uracilresidue is considered to be identical to a thymine residue and whenassessing complementarity a uracil residue is considered to becomplementary to/capable of forming hydrogen bonds with an adenineresidue.

The determination of the degree of complementarity of two or moresequences can be performed by any method known in the art. Preferably,the method used is that set out in Hossbach et al. (supra). Inaccordance with this method, the Perl script accessible athttp://www.mpibpc.mpq.de/croups/luehrmann/siRNA is used.

In addition, various tools for the design and analysis of short RNAmolecules are well-known, which permit one of ordinary skill in the artto determine those RNA molecules which can achieve effective andspecific down-regulation of a target RNA transcript. Established methodsinclude, for example, the GPboost and Reynolds algorithms (PMIDs:15201190, 14758366). In addition, the ability of a short RNA to causeeffective down-regulation of a target RNA can be evaluated usingstandard techniques for measuring the levels of RNA or protein in cells.For example, a short RNA of the invention can be delivered to culturedcells, and the levels of target RNA can be measured by techniquesincluding but not limited to Northern blot or dot blotting techniques,or by quantitative RT-PCR.

Preferably the short RNAs possess none of the motifs aaaa, cccc, gggg,or uuuu. Preferably the short RNAs have a GC-percentage of at least 20%and no more than 75%, i.e. between 20% and 75%, preferably between 20%and 55%. The short RNAs of the above methods are ideallythermodynamically stable duplexes, in which case the GC percentage ofeach strand is at least 25% and no more than 75%, i.e. between 25% and75%, preferably between 20% and 55%.

Tools and algorithms for determining whether or not RNAs possess themotifs aaaa, cccc, gggg or uuuu and for determining the percentage GCcontent of the molecules/strands are well known to the skilled artisan.Such tools include those described and referenced in Saetrom and Snove,(2004) Biochem Biophys Res Commun 321: 247-253 and Vert et al., (2006)BMC Bioinformatics 7: 520 (17 pages).

Short RNAs can induce down-regulation of non-target transcripts thathave a limited number of mismatches to the short RNA strand which isincorporated into the RISC protein complex. This reduces the efficiencyof the short RNA molecule and is therefore not desired. Consequently,short RNA molecules should have limited complementarity to transcriptsother than the intended target to prevent unintended off-target effects.The probability of a short RNA candidate having cleavage-basedoff-target effects is a function of its complementarity to non-targetRNA sequences and can be determined by any known method in the art.Optionally, an ungapped Smith-Waterman method (TF Smith & MS Waterman(1981) Journal of molecular biology 147: 195-197) can be used to screenthe candidate short RNA against the Ensembl (Flicek, P., et al. (2008)Ensembl 2008. Nucleic Acids Res 36: D 707-714) human transcriptomedatabase (Snøve, O., Jr., et al. (2004) Biochem Biophys Res Commun 325:769-773) to identify a short RNA's potential off-target transcripts.Alternatively, the short RNA can be screened against a population ofchosen RNA sequences, for example a selection of GenBank sequences,which do not encompass the entire Ensembl human transcriptome database.Alternatively, a Hamming distance measure can be used.

Preferably, the short RNA molecules have more than two mismatches to theidentified off-target transcripts. Alternatively viewed, preferably theshort RNA molecules have a Hamming distance of 2 or greater to allpotential off-target transcripts. If the short RNA is double strandedthen preferably both strands satisfy this requirement.

Optionally, the short RNA molecules have characteristics in common withknown highly effective standard siRNAs. Preferably, the short RNA, or ifdouble stranded one or both strands of the short RNA, has a GPboostscore of more than 0.1. GPboost is a known genetic programming-basedprediction system of siRNA efficacy and the methods used for determiningthe GPboost score of siRNA strands is disclosed in “Predicting theefficacy of short oligonucleotides in antisense and RNAi experimentswith boosted genetic programming”, Pal Saetrom (2004) Bioinformatics20(17): 3055-3063, the content of which is incorporated here byreference. Alternatively or in addition, the short RNA molecules possessspecific sequence features which are associated with highly effectivesiRNAs. The algorithm described by Reynolds [Reynolds et al. (2004)Nature biotechnology 22(3):326-330], which is incorporated here byreference permits the determination of whether or not short RNAs possesssufficient features of this type. One of ordinary skill in the art wouldbe able to define and refine his threshold for his particular purpose.

Optionally, the short RNA molecules contain position-specific sequencemotifs which are associated with highly effective siRNAs. siRNA efficacyprediction algorithms are well-known in the art and motifs which areassociated with highly-effective siRNAs are discussed in Saetrom andSnove, (2004) Biochem Biophys Res Commun 321: 247-253, the content ofwhich is incorporated here by reference.

Preferably the short RNA molecule is capable of direct entry into theRNAi machinery of a cell or is capable of being processed by Dicerbefore entry into the RNAi machinery of a cell. Methods of determiningwhether or not a short RNA molecule is capable of being processed byDicer before entry into the RNAi machinery of a cell are well-known inthe art, for instance in vitro Dicer assays such as that disclosed inTiemann et al. (2010) RNA 16(6): 1275-1284 and Rose et al. (2005)Nucleic Acid Research 33(13):4140-4156.

If the short RNA molecule is double stranded and if only one strandwithin the molecule is capable of effectively and specificallydown-regulating the target RNA transcript then preferably that strand ispreferentially loaded into RISC. The design of double-stranded RNAmolecules in which one strand is preferentially loaded into RISC iswithin the competence of one of ordinary skill in the art. For instance,the 5′ end of the strand of the short RNA molecule which targets thetarget RNA transcript can be made or selected to be lessthermodynamically stable than the 5′ end of the other strand. Preferablythere is a large difference in duplex thermodynamic end stability suchthat the 5′ end of the strand of the short RNA molecule which targetsthe target RNA transcript is less thermodynamically stable than the 5′end of the other strand. The absolute value of the difference in duplexthermodynamic end stability (ΔΔG) can be calculated in accordance withany method standard in the art. Optionally, the absolute value of thedifference in duplex thermodynamic end stability is calculated byRNAfold (Hofacker et al., (2003) Nucleic Acids Research Vol. 31, No. 13,pp 3429-3431) by considering the 5 closing nucleotides at the ends ofthe duplex. Preferably the absolute value of the difference in duplexthermodynamic end stability as calculated by RNAfold is more than 0kcal/mol, more preferably more than 1 kcal/mol, more preferably morethan 3 kcal/mol.

Many standard tools for short RNA design, such as those described above,provide means for assessing this property of the molecules. Forinstance, double-stranded molecules can be selected if they havethermodynamic properties which favour the incorporation of one strandover the other into the RNAi machinery. Alternatively, the preferentialloading of one strand can be achieved by using dsRNAs which contain RNAthat differs from naturally-occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides.

Methods of determining the target RNA transcripts present in a cell arewell-known in the art. For instance, the genomic region around the locusof the gene of interest can be searched for spliced expresses sequencetags. An expressed sequence tag or EST is a short sub-sequence of atranscribed cDNA sequence. ESTs are commonly used to identify genetranscripts. Public databases of ESTs are known in the art, for instancethe GenBank database. Alternatively, Reverse Transcriptase PCR (RT-PCR),a well-known tool for identifying RNA, can be used to identify potentialtarget RNA transcripts. Alternatively, high throughput sequencing orother such methods can be used to sequence total, size-fractionated, orother suitable subsets of RNAs and use such sequencing libraries toidentify RNA transcripts that originate from the region of interest.Alternatively, a population of known RNA transcripts can be searched toidentify suitable transcripts. Any database of RNA transcripts known inthe art can be used, for instance the University of California SantaCruz (UCSC) Spliced EST track. Alternatively, the population may beprepared from a population possessed by the skilled man working theinvention for his own specific purposes. For instance, if the targetgene is known to be expressed in a particular cell type, then thedatabase of transcripts may be those which have been determined to bepresent in that cell type. The skilled man will be able to determine thepopulation to use for his specific desired purposes.

However, for the purpose of the present invention the positiveidentification of any RNA transcripts which are antisense to the targetgene is not in fact required. Thus, the existence of said non-coding RNAtranscript (i.e. the target transcript to be down-regulated) need not bedetermined. The present inventor found that if the nucleotide sequenceof the coding strand of the gene in the region surrounding the gene'stranscription start site is obtained, i.e. determined by sequencing orfound on a database, and the reverse complementary RNA sequence to thatregion is determined, then short RNA molecules which are complementaryto that latter sequence can be used to up-regulate the target gene.Complementarity requirements are discussed elsewhere herein. The regionsurrounding the gene's transcription start site is the region locatedbetween 100, 200, 300, 400, 500, 800, 1000 or 2000 nucleotides upstreamand downstream of the transcription start site.

Thus, the saRNA molecules may be designed based on the principles setout above. In some embodiments, the approach described below may betaken.

For example, FIG. 4 shows a block diagram of a method 400. In the method400, a selection block 410 includes selecting a gene sequence of a genefor purposes of designing one or more short activating RNA molecules.Preferred genes are discussed elsehwere herein. In a provision block420, the method 400 includes providing information such as informationabout a target genomic location, orientation and transcriptionalstructure (e.g., from a database). In an identification block 430,identification of one or more antisense transcripts occurs (e.g.,searching data for transcripts that are antisense to and in the vicinityof the target gene), although it not necessary to actually determine theexistence of a non-coding RNA transcript. In a provision block 440, themethod 400 includes providing a bounded region about a transcriptionstart site (TSS). For example, a number of units on one side and anumber units on another side of a TSS may be provided as a boundedregion. In a design process commencement block 450, the method 400 maycommence a computer-aided process that acts to facilitate to design oneor more saRNAs. Such a process may rely on one or more techniquesselected from, for example, statistical techniques, genetic programming(GP) techniques, boosting techniques, neural network techniques, hiddenMarkov model (HMM) techniques, vector machine (SVM) techniques, etc. Ingeneral, such techniques provide for designing one or more saRNAs whenprovided a bounded TSS region as an input.

In the example of FIG. 4, the method 400 also includes an output block460, which can output one or more designed saRNAs and optionally callfor or actively take one or more steps to construct one or more saRNAs.As to output, the block 460 may output information to a display, aprinter, memory, an interface, etc. For example, the block 460 mayoutput information to a network via a network interface, outputinformation to a machine configured for chemical processing to constructmolecules, etc.

Also shown in FIG. 4 are various devices 412, 422, 432, 442, 452 and462, which may be storage media configured to store instructions forperforming one or more actions of the associated blocks 410, 420, 430,440, 450 and 460. For example, the blocks 412, 422, 432, 442, 452 and462 may be one or more computer-readable media that include informationsuch as processor- or computer-executable instructions. While shownindividually, a single storage medium may include instructions for morethan one of the blocks 412, 422, 432, 442, 452 and 462. A storage mediummay be a hard drive, an optical disk, memory (e.g., a memory card), etc.

As described herein, one or more computer-readable media can includecomputer-executable instructions to instruct a computing system toreceive information (e.g., as in block 420), to identify antisensetranscripts (e.g., as in block 430), to identify a bounded region for atranscription start site (e.g., as in block 440) and to call forcommencement of a saRNA design process (e.g., as in block 450). Such oneor more computer-readable media may optionally include instructions toinstruct a computing system to design one or more saRNAs. Further, oneor more computer-readable media may include instructions to cause acomputing system to write or transmit one or more designed saRNAs. Forexample, information about one or more saRNAs may be written to astorage medium, transmitted via an interface (e.g., a networkinterface), etc. Accordingly, a machine configured for constructing oneor more saRNAs may receive such information and subsequently constructsuch one or more saRNAs (e.g., as in block 460). As another example,consider a scenario where one or more saRNAs are available from one ormore sources. As described herein, a search of such one or more sourcesmay occur to identify one or more available saRNAs (e.g., consider acatalog, a saRNA bank, etc.).

As described herein, in various trials, the gene sequence for humanalbumin was selected (see, e.g., block 410). Such a gene sequence wasselected for designing short activating RNA molecules for its specificactivation where four parameters were used: 1) targeting geneannotations from UCSC RefSeq database; 2) targeted sequence fromantisense RNA; 3) promoter selection of antisense sequences; and 4)identification of candidate short activating RNAs. Next, downloading ofinformation occurred for the target's genomic location, orientation, andtranscriptional structure from one or more available databases (e.g.,RefSeq at UCSC) (see, e.g., block 420).

Next, given the database of RNA transcripts with known read direction,such as the UCSC Spliced EST track, searching the database occurred fortranscripts that are antisense to and in the vicinity of the target gene(see, e.g., block 430). More specifically, a process includedidentifying antisense transcripts that (a) overlap the target's promoterand the target mRNA's 5′ end; (b) overlap the target mRNA; (c) are atmost 20-100kb upstream of the target's transcription start site (TSS);or (d) are at most 20-100 kb downstream of the target's poly-adenylationsite. A process can use these four criteria as hierarchical filters suchthat if it finds antisense transcripts that for example satisfycriterion (a), the process does not necessarily need to consider thethree other criteria.

Next, based on the target's transcription start site (TSS), a processcan include downloading the antisense genomic sequence from, forexample, a fixed size region upstream and downstream of the TSS. Invarious trials, a typical region size used was 500 nts upstream anddownstream of TSS, but larger or smaller sizes may be suitablyimplemented (see, e.g., block 440).

Following the downloading, a process may include designing one or moresiRNAs that give effective and specific down-regulation of the antisensetarget sequence (see, e.g., block 450). For example, a process may (a)use a siRNA design algorithm, such as GPboost, to identify candidateeffective siRNAs; (b) remove all candidate siRNAs with aaaa, cccc, gggg,or uuuu motifs and GC content less than 20% or greater than 55%; (c)remove all candidates that have Hamming distance less than two to allpotential off-target transcripts; and (d) return a given number ofremaining non-overlapping siRNAs sorted by their predicted siRNAknockdown efficacy. In such an example, the process can return a number(e.g., the two highest scoring) of saRNAs for a given antisense targetsequence. As described herein, output and construction of one or moresaRNA followed (see, e.g., block 460).

The saRNAs of the invention can be produced by any suitable method, forexample synthetically or by expression in cells using standard molecularbiology techniques which are well-known to the skilled artisan. Forexample, the saRNAs can be chemically synthesized or recombinantlyproduced using methods known in the art.

As mentioned above, the saRNAs and/or cells may be used therapeutically.The saRNAs or cells may be administered via injection, e.g.intravenously, subcutaneously, intramuscular or into a target organ.Thus, injection may be systemic or at or into the target site, e.g. atarget organ, preferably the liver, although the prostate or pancreas isalso contemplated. Alternatively, administration may be oral or pr (perrectum). Injection of a cell into the liver is preferred.

The short RNAs and/or cells of the invention may be administered to apatient in need thereof by any means or delivery vehicle known in theart, for example via nanoparticles, cationic lipids, lipids such ascholesterol or a-tocopherol, liposomes, e.g. positively charged cationicliposomes, polymers, such as polyethyleneimine, dendrimers, aptamers, oras antibody conjugates. The short RNAs may also be administered as viralvector expressed shRNAs or miRNA mimics.

Preferably, the saRNA or cell is associated, e.g. complexed with, linkedto, or contained within, a moiety that targets the saRNA or cell to aspecific tissue or cell type, e.g. liver cells, e.g. hepatocytes. Saidmoiety may be one of the means/delivery vehicles mentioned above.However, targeted delivery may not in fact be required, because drugsand particularly saRNAs are naturally delivered to the liver followingsystemic administration.

Aptamers are oligonucleotides or peptides with high selectivity,affinity and stability. They assume specific and stablethree-dimensional shapes, thereby providing highly specific, tightbinding to target molecules. For any specific molecular target, nucleicacid aptamers can be identified from combinatorial libraries of nucleicacids, e.g. by a technique called systematic evolution of ligands byexponential enrichment (SELEX) (see, for example, Tuerk C and Gold L:Systematic evolution of ligands by exponential enrichment: RNA ligandsto bacteriophage T4 DNA polymerase. Science 1990, 249:505-510.). Peptideaptamers may be identified e.g. using a yeast two hybrid system. Theskilled person is therefore able to design suitable aptamers fordelivering the saRNAs or cells of the present invention to target cellssuch as liver cells. DNA aptamers, RNA aptamers and peptide aptamers arecontemplated. Administration of short RNAs of the invention to the liverusing liver-specific aptamers is particularly preferred.

Also provided is a conjugate of an aptamer and an short RNA of theinvention. The conjugate may be formed using any known method forlinking two moieties, such as direct chemical bond formation, linkagevia a linker such as streptavidin and so on.

Methods of generating antibodies against a target cell surface receptorare well known. The saRNA molecules of the invention may be attached tosuch antibodies, for example using RNA carrier proteins. The resultingcomplex may then be administered to a subject and taken up by the targetcells via receptor-mediated endocytosis. The cells of the invention maybe linked to such antibodies using known means.

The saRNA or cells may be encapsulated in liposomes using methods knownin the art. The liposomes may optionally be associated with atarget-cell specific moiety such as an antibody or a peptide.

As discussed above, the molecules of the present invention are generatedbased on sequence analysis of a target gene and methods and productsassociated with delivery of these short nucleic acid molecules arefurther aspects of the invention. Thus, the present invention alsoprovides methods and products, including computer-readable storage mediaand data structures, which may be set out schematically in FIG. 4-FIG. 9and are discussed below with reference to FIG. 4-FIG. 9. Unlessotherwise stated, the definitions, description and preferred embodimentsdescribed above in relation to the methods and products of the presentinvention apply, mutatis mutandis, to the aspects discussed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following examples are intended to be illustrative of the presentinvention and to teach one of ordinary skill in the art to make and usethe invention. These examples are not intended to limit the invention inany way. The invention will now be further described in the followingExamples and the Tables and Figures.

A more complete understanding of the various methods, devices,assemblies, systems, arrangements, etc., described herein, andequivalents thereof, may be had by reference to the following detaileddescription when taken in conjunction with examples shown in theaccompanying drawings where:

FIG. 1 is a diagram of some possible mechanisms (Mechanism A andMechanism B);

FIG. 2A and FIG. 2B are a series of plots of results for saRNAtransfected HepG2 cells demonstrating upregulation of albumin. See Table1 for the sequences of albumin PR1, PR2, PR3 and PR4;

FIG. 3A and FIG. 3B are a series of plots of for WST-1 proliferationassay results in HepG2 cells following saRNA transfection. See Table 1for the sequences of albumin PR1, PR2, PR3 and PR4;

FIG. 4 is a block diagram of an example of a method;

FIG. 5 is a block diagram of examples of equipment and a computingdevice;

FIG. 6 is a block diagram of an example of a method;

FIG. 7 is a block diagram of an example of a method;

FIG. 8 is a block diagram of an example of a treatment plan and otherexamples of techniques, technologies, etc;

FIG. 9 is a diagram of saRNA being delivered to one or more types ofcells.

FIG. 10A-10C shows the in vitro effects of transfecting liver cells withalbumin saRNA (see Example 2). Data represent mean, SEM from threeindependent transfections. FIG. 10A: mouse albumin ELISA resultscomparing HepG2 cells transfected with saRNA specific to albumin againstcontrol HepG2 cells. *=p(0.0136). FIG. 10B: qPCR analysis of albuminmRNA, comparing HepG2 cells transfected with saRNA specific to albuminagainst control HepG2 cells. **=p(<0.003). FIG. 10C: qPCR analysis ofalbumin mRNA, comparing rat liver epithelial cells transfected withsaRNA specific to albumin against control rat liver epithelial cells.

FIG. 11A-11D show the effects of transfecting the HepG2 cell line withCEBPA saRNA constructs AW1 and AW2 against control (see Example 4). FIG.11A shows the relative mRNA transcript levels of CEBPA, FIG. 11B showsthe relative mRNA transcript levels of albumin, FIG. 11C shows albuminexpression (ng/ml) and FIG. 11D shows HepG2 cell viability.

FIG. 12A-12C show the effects of transfecting the DU145 prostrate cancerepithelial cell line with CEBPA saRNA constructs AW1 and AW2 againstcontrol (see Example 4). FIG. 12A shows the relative mRNA transcriptlevels of CEBPA, FIG. 12B shows the relative mRNA transcript levels ofalbumin and FIG. 12C shows HepG2 cell viability.

FIG. 13A-13D show blood analysis of tail injected mice (n=5) for eachcontrol, or dendrimer+albumin saRNA group (see Example 5) FIG. 13AAlbumin, FIG. 13B gamma glutamyl transpeptidase, FIG. 13C alanineaminotransferase and FIG. 13D aspartate aminotransferase.

FIG. 14 shows the serum albumin levels after the administration of AW1saRNA to rats, compared against control. See Example 6. AW1 targetsCEBPA (see Table 1 for sequences).

FIG. 15 shows the pooled data regarding serum albumin levels, combiningdata of AW1 and AW2 (see Table 1 for sequences) administration to ratsand comparing against control administration to rats. See Example 6.

FIG. 16A and FIG. 16B show quantitative analysis of transcript levelfrom tissue biopsies in mice administered with albumin saRNA + dendrimeragainst control. FIG. 16A shows the relative mRNA transcript levels of αfeto protein (AFP) and FIG. 16B shows the relative mRNA transcriptlevels of hepatocyte growth factor (HGF).

Table 1 shows short RNA molecules designed for upregulating albuminexpression.

Table 2 shows short RNA molecules designed for upregulating albuminexpression.

More specifically, FIG. 2A-2B show a plot of data as to saRNAtransfected HepG2 cells to demonstrate upregulation of albumin. In FIG.2A represents a plot of results from HepG2 cells that were plated at adensity of 2.5×10⁵ cells/well in a 24 well plate. Four clones of saRNAwere directed at the promoter regions (PR1, PR2, PR3 and PR4) of thealbumin gene were used. Cells were transfected with 150 ng of saRNA at0, 12 and 24 hours following plating in a 24 well plate followed byharvesting for extraction of total RNA. An RT-PCR profile of the mRNAlevels showed an increase in albumin levels only in cells transfectedwith saRNAs. In FIG. 2B represents a plot of results from asemi-quantitative analysis from two independent trials to show that PR3saRNA had the most marked increase in albumin mRNA level (158%+/5.7%).As to FIG. 3, results from a WST-1 proliferation assay are shown inHepG2 cells following saRNA transfection. HepG2 cells were plated at adensity of 1.5×10⁵ cells/well in a 24 well followed by threetransfections at 0, 12 and 24 hours. WST-1 reagent was then added for 30minutes before analysis in a multiplate reader at Amax450 nm. In FIG. 3Ais a plot that presents the amount of formazan dye, indicative ofmetabolically active and proliferative cells, which was drasticallyreduced only in cells that were transfected with saRNA to albumin. InFIG. 3B is a plot that presents the percentage of cell viabilityrelative to untransfected cells to demonstrate that cells transfectedwith PR3 saRNA had the most marked decrease in cell proliferation.

As to details of various trials described herein, specifically as tocell culture, HepG2 cells (American Type Culture Collection) werecultured in RPMI-140 media (Sigma, USA) supplement with 10% fetal calfserum (FCS) (Invitrogen, USA), 100 units/ml penicillin, 0.1 mg/mlstreptomycin, 2 mmol/L glutamine (Sigma, USA) at 37° C. in humidified 5%CO2 air.

As to chemical processing, for various trials, paired saRNAoligonucleotides were annealed using 50 mM Tris-HCl, pH8.0, 100 mM NaCland 5 mM EDTA following a denaturation step at 90° C. followed by agradual anneal step to room temperature.

A process of isolation of total RNA for semiquantitative rtPCR caninclude various acts. For example, all total RNA extraction can becarried out using the RNAqueous-Micro kit (Ambion, UK) (e.g., followingthe manufacturer's instructions). As to various trials, cells weregently centrifuged followed by 3 pulses of sonication at Output 3 inLysis buffer (Ambion, UK). The cell lysates were then processed throughan RNA binding column, followed by multiple washes and elution. Thetotal RNA isolated was quantified by a Nanodrop 2000 spectrophotometer.500 ng of total RNA was reversed transcribed using One Step RT-PCR(Qiagen, Germany) (e.g., following the manufacturer's instructions).Expression for albumin and for loading control, a house keeping geneactin was performed by PCR using their respective primer pairs:albumin-F: TCC AGC ACT GCC TGC GGT GA; R: TCC GTC ACG CAC TGG GAG GA;following 37 cycles at 95° C.-45 sec; 55° C.-45 sec; 61° C.-45 sec.Actin-F: GAG AAA ATC TGG CAC CAC ACC; R: ATA CCC CTC GTA GAT GGG CACfollowing 37 cycles at 95° C.-5 min; 60° C.-30 sec; 70° C.-45 sec; 72°C.-10 min at 37 cycles. Products were analyzed in triplicate semiquantitatively using UVP VisonWorks LS (v6.2.).

For trials described herein, the tetrazolium salt,4-[3-(4-iodophenyl)2-(4nitrophenyl)2H-5-tetrazolio]1,3benzenedisulfonate (WST-1)-proliferation assay was used. Specifically, HepG2(1.5×10⁵ cells/well) were plated in a 96 well plate and cultured in 500ul of RPMI-140 media (Sigma, USA) supplement with 10% fetal calf serum(FCS) (Invitrogen, USA), 100 units/ml penicillin, 0.1 mg/mlstreptomycin, 2 mmol/L glutamine (Sigma, USA) were transfected usingNanofectin (transfection of 150 ng of annealed saRNA targeted to MafAusing Nanofectamine (PAA, UK) (e.g., following the manufacturer'sinstructions). The foregoing process was repeated three times at 0 hr,12 hr and 24 hr from plating. The cell proliferation reagent WST-1(Roche Applied Science, UK) was added (e.g., following themanufacturer's instructions). The colorimetric assay was then incubatedfor 30 minutes to allow cleavage of the tetrazolium salt WST-1 to aformazan dye by mitochondrial succinate-tetrazolium reductase in viablecells. The quantity of formazan dye, which is directly related to thenumber of metabolically active cells proliferating was measured atAmax450 nm in a multiwall plate reader. For trials, a total of threeindependent experiments were assayed.

Again, as shown in FIGS. 2A-2D and 3A-3B, saRNA transfected HepG2 cellsdemonstrated upregulation of albumin and WST-1 proliferation assayresults, in HepG2 cells following saRNA transfection, demonstratedreduced proliferation.

FIG. 5 shows a system 500 (e.g., a computing system) that may includevarious components. In the example of FIG. 5, the system 500 may includeinput information 510, which may be provided to processor(s) 520 andmemory 530, for example, via a network interface 540 or other interface.As shown, a CRM block 534 may include one or more computer-readablemedia. Such medium or media may be one or more of those of FIG. 4 (see,e.g., 412, 422, 432, 442, 452 and 462). Also shown in FIG. 5 is adisplay device 550, a storage device 570 and a manufacturing device 590.

As described herein, the manufacturing device 590 may include one ormore chemicals (e.g., one or more probes, optionally one or more otherchemicals such as an enzyme, etc.) and optionally vessels, wells, etc.,for holding one or more chemicals.

In the example of FIG. 5, the system 500 can include one or moreprocessors 520 operatively coupled to memory 530, which may beconfigured to store instructions read from one or more computer-readable(or processor-readable) storage media 534. As described herein, the CRM534 may store instructions that, upon execution by the one or moreprocessors instruct the system 500 to perform at least part of themethod 400 of FIG. 4.

Also shown in the example of FIG. 5 is the network interface 540, whichmay allow for communication of information to or from the system 500.For example, instructions may be communicated to the system 500 (e.g.,to commence operation, terminate operation, quality control, updatesoftware instructions, etc.). Output of the processor(s) 520 may becommunicated from the system 500 via the network interface 540 (e.g., toa healthcare provider, a scientist, a database, a manufacturer ofpharmaceuticals, etc.).

FIG. 6 shows a block diagram of a method 600. As mentioned, saRNA may beuseful for treatment or prevention of cancer, such as primary livercancer. For example, as to liver cancer, treatment or prevention mayoccur by administering saRNA to patients with liver cirrhosis (e.g., dueto viral hepatitis or ethanol intoxication). The method 600 includes adiagnosis block 610 for diagnosing a patient condition, a provisionblock 620 for providing saRNA and an administration block 630 foradministering the provided saRNA to the patient. As described herein,such actions may occur, wholly or partially, via a machine or machines,optionally operating based on instructions such as instructions storedon one or more computer-readable media (see, e.g., CRM 612, 622 and632).

The method 600 of FIG. 6 may be performed in conjunction with one ormore block of the method 400 of FIG. 4. For example, the provision block620 may include designing as described with respect to the block 450 ofthe method 400 FIG. 4.

As to the diagnosis block 610, it may provide for diagnosing cancerouscells 611, diagnosing pre-cancerous cells 613, diagnosing other risk 615or any combination thereof. As mentioned, certain conditions pose risksfor cancer. For example, patient infections, patient habits, patientintoxication, etc., can pose risks for cancer. As described herein, amethod can include inputting information, assessing risk based at leastin part on the information and deciding whether to administer RNA to apatient or patients similarly situated. Such administration (e.g., oradministrations) may be, for example, for prevention of cancer ortreatment of cancer.

FIG. 7 shows a block diagram of a method 700. In a provision block 710,a saRNA is provided, optionally with a carrier. For example, a designedsaRNA may optionally be carried with a liposome or an aptamer or an RNAaptamer to a liver cancer such as a liver cancer that has metastasizedoutside the liver. In an administration block 720, the saRNA (andoptionally carrier) is administered to a patient (e.g., a humansubject). As an example, for liver tumours, a designed saRNA may beadministered to reach the liver of a patient by systemic injection orlocal by percutaneous direct injection. Whether for a liver tumour orother type of tumour, administration may be guided by a guidancetechnique per block 725. Guidance techniques may include ultrasound,fluoroscopy, MR, CT, laparotomo or laparoscopy of endovascular delivery,etc. As described herein, administration may be via oral delivery.

In the example of FIG. 7, an additional therapy may be provided, asindicated by a decision block 730. An additional therapy may optionallybe selected from one or more of the following therapies RF ablation,microwave ablation, selective radiotherapy, irreversibleelectroporation, high-focused ultrasound, transarterialchemoembolization, etc. As indicated, if the decision block 730 decidesthat one or more additional therapies are to be delivered, then themethod 700 enters an additional therapy block 740. If the decision block730 decides that no additional therapy is to be delivered, the method700 enters an optional wait block 735 and then continues at theadministration block 720, as appropriate. For example, for a therapythat administers saRNA to a patient (e.g., in other than a zero-orderfashion), a dose may diminish in effectiveness over a period of days.Thus, the wait block 735 may provide a wait time before administrationof a subsequent dose. As an example, where doses are administered, adose may be given to a patient several times a week (e.g., two to threedoses a week for prevention or treatment). As described herein, one ormore additional therapies may present factors that determine or alter await time between successive doses of saRNA.

Also shown in FIG. 7 are various devices 712, 722, 727, 732, 737 and742, which may be storage media configured to store instructions forperforming one or more actions of the associated blocks. For example,these may be one or more computer-readable media that includeinformation such as processor- or computer-executable instructions.While shown individually, a single storage medium may includeinstructions for more than one of the blocks. A storage medium may be ahard drive, an optical disk, memory (e.g., a memory card), etc.

FIG. 7 also shows a treatment planning module 770. In the example ofFIG. 7, the module 770 includes a dynamics for RNA block 772, a dynamicsfor chemotherapy block 774, a dynamics for radiation therapy block 776,a patient information block 778, a determinations block 780 and anoutput block 782. As described herein, RNA, as administered, may haveparticular dynamics and one or more other therapies may have particulardynamics. As described herein, opportunities exist for synergies betweenan RNA therapy (e.g., saRNA) and one or more other therapies. Thetreatment planning module 770 of FIG. 7 may be part of a computingsystem (e.g., in the form of computer-executable instructions) thatallows for automatic or interactive planning of treatment for a patientor patients.

FIG. 8 shows the treatment planning module 770 of FIG. 7, which may bemade of multiple sub-modules, along with an example of a treatment plan810 displayed (e.g., rendered) graphically as a graphical user interface(GUI) 811. Accordingly, the sub-module 782 may include instructions toforming a GUI that provides for input by a user to interact with theplanning module 770. For example, a user may be able to select or adjustdose and types of therapies to optimize a treatment plan for a patient(see, e.g., saRNA doses given between chemo doses). Kinetics of action,clearance, etc., may be taken into account during planning (e.g., asprovided by a dynamics sub-module).

FIG. 8 also shows a RNA dose 812, which may be saRNA 813 or mRNA 815. Asto the mRNA dose 815, this may be provided with an aptamer 816 or withgene therapy 817, for example, to cause upregulation of a polypeptide(e.g., albumin or other polypeptide). FIG. 8 also shows a chemo dose 822as an example, which may be chemotherapy suited for slow proliferationcells 823 or chemotherapy suited for fast proliferation cells 825.Factors such as cell cycle time, S-phase duration, etc., have beenreported as being germane to effectiveness of chemotherapy. Moreparticularly, cell proliferation kinetics can be a factor as to overalleffectiveness of chemotherapy.

As described herein, a dose or doses of RNA (e.g., saRNA) may beadministered to a patient (e.g., to tumor cells, etc.) as a mechanism todecrease proliferation after, before or after and before administrationof chemotherapy. As chemotherapy may be delivered in spread out doses(e.g, due to toxicity risk to a patient), administration of saRNA in aperiod between chemo doses may act to optimize overall treatment of apatient.

As described herein, a method can include administering saRNA to cause acell to upregulate production of a polypeptide (e.g., “naturallysecreted”) by the cell before it turns cancerous or pre-cancerous. Forexample, albumin is a polypeptide naturally secreted by various cells(e.g., before such cells turn cancerous or pre-cancerous). As describedherein, a method can include administering saRNA to cause anundifferentiated cell to revert to a differentiated cell (e.g., to shiftcell behavior of a cell towards behavior more characteristic of adifferentiated cell).

FIG. 9 shows a diagram of saRNA being delivered to a normal cell, aprecancerous cell and a cancerous cell along with illustrative plots ofrate of molecule production and rate of cell proliferation. Asindicated, saRNA can be delivered to one or more types of cells, causeincrease in rate of production of a molecule and cause decrease in rateof cell proliferation.

As described herein, short activating RNA can include a sequence ofunits to cause upregulation of a polypeptide of a mammalian cell whereproduction of the polypeptide by the mammalian cell impacts negativelyproliferation capability of the mammalian cell. As described herein, apolypeptide (as associated with saRNA) may be a naturally secretedpolypeptide of a mammalian cell in its natural state. As describedherein, a polypeptide (as associated with sa RNA) may be albumin.

As described herein, saRNA may be provided or designed or designed andprovided for a hepatocyte. More generally, saRNA may be provided ordesigned or designed and provided for a normal cell, a precancerouscell, a cancerous cell where such cells may be mammalian cells.

As described herein, a method can include diagnosing a patient with acondition characterized by excessive proliferation of a type of cell;and designing a short activating RNA to upregulate production of apolypeptide by the type of cell wherein the upregulated production ofthe polypeptide impacts negatively proliferation capability of the typeof cell. Such a method may further include producing the shortactivating RNA. Such a method may further include administering thedesigned short activating RNA to the patient. As to diagnosing, a methodmay include diagnosing liver cancer. As mentioned, a method can includeadministering an additional therapy (e.g., non-saRNA therapy) thattargets a type of cell associated with a saRNA therapy. Such anadditional therapy may be chemotherapy, radiation therapy, RF ablationtherapy, microwave ablation therapy or other therapy.

As described herein, a method can include designing saRNA by executingone or more instructions stored on a computer-readable storage mediumresponsive to providing a transcription start site for a gene having acoding region for the polypeptide.

As described herein, one or more computer-readable media can includecomputer-executable instructions to instruct a computing system to:receive a transcription start site for a gene having a coding region foralbumin; select a bounded region about the transcription start site;characterize strings of units as to saRNA candidates for upregulatingproduction of albumin; and output one or more characterized strings ofunits as preferred candidates for manufacture of saRNA molecules foradministration to a human subject to treat liver cancer.

Further embodiments of the invention are set out below:

1. Short activating RNA comprising: a sequence of units to causeupregulation of a polypeptide of a mammalian cell wherein production ofthe polypeptide by the mammalian cell impacts negatively proliferationcapability of the mammalian cell.

2. The short activating RNA of embodiment 1 wherein the polypeptidecomprises a naturally secreted polypeptide of the mammalian cell in itsnatural state.

3. The short activating RNA of embodiment 1 wherein the polypeptidecomprises albumin.

4. The short activating RNA of embodiment 1 wherein the mammalian cellcomprises a hepatocyte.

5. The short activating RNA of embodiment 1 wherein the mammalian cellcomprises a cancer cell.

6. A method comprising: diagnosing a patient with a conditioncharacterized by excessive proliferation of a type of cell; anddesigning a short activating RNA to upregulate production of apolypeptide by the type of cell wherein the upregulated production ofthe polypeptide impacts negatively proliferation capability of the typeof cell.

7. The method of embodiment 6 further comprising producing the shortactivating RNA.

8. The method of claim 6 further comprising administering the designedshort activating RNA to the patient.

9. The method of embodiment 6 wherein the diagnosing diagnoses livercancer.

10. The method of embodiment 6 wherein the designing comprises executingone or more instructions stored on a computer-readable storage mediumresponsive to providing a transcription start site for a gene having acoding region for the polypeptide.

11. The method of embodiment 6 further comprising administering anadditional therapy that targets the type of cell.

12. The method of embodiment 11 wherein the additional therapy comprisesa therapy selected from a group consisting of chemotherapy, radiationtherapy, RF ablation therapy, and microwave ablation therapy.

13. One or more computer-readable media comprising computer-executableinstructions to instruct a computing system to: receive a transcriptionstart site for a gene having a coding region for albumin; select abounded region about the transcription start site; characterize stringsof units as to saRNA candidates for upregulating production of albumin;and output one or more characterized strings of units as preferredcandidates for manufacture of saRNA molecules for administration to ahuman subject to treat liver cancer.

14. A short activating RNA includes a sequence of units to causeup-regulation of a polypeptide of a mammalian cell where production ofthe polypeptide by the mammalian cell impacts negatively proliferationcapability of the mammalian cell.

Various other examples of technologies, techniques, devices, assemblies,systems, methods, etc., are also disclosed.

Although some examples of methods, devices, systems, arrangements, etc.,have been illustrated in the accompanying Drawings and described in theforegoing Detailed Description, it will be understood that the exampleembodiments disclosed are not limiting, but are capable of numerousrearrangements, modifications and substitutions without departing fromthe spirit set forth and defined by the following claims.

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Tables

TABLE 1 SEQ SEQ ID ID ID Target (Sense) NO Anti-sense (Guide) NO humanPR1 CCUUGUAAGACUUCACAAA  5 UUUGUGAAGUCUUACAAGG  6 albumin PR2UGGAUAGGUCUUUGGGAUA  7 UAUCCCAAAGACCUAUCCA  8 PR3 AAGAGUUAAGUCCCAAAUU  9AAUUUGGGACUUAACUCUU 10 PR4 GACUAAAUCCCUUGUGUAU 11 AUACACAAGGGAUUUAGUC 12human AW1 CGGUCAUUGUCACUGGUCA 13 UGACCAGUGACAAUGACCG 14 rCEBPra AW2AGCUGAAAGGAUUCAUCCU 15 AGGAUGAAUCCUUCCAGCU 16 NR1 ACAUAGUCCCAGUGAUUAA 17UUAAUCACUGGGACUAUGU 18 NR2 GAAUAAGACUUUGUCCAAU 19 AUUGGACAAAGUCUUAUUC 20human PR1 GCGCGGAUUCUCUUUCAAA 21 UUUGAAAGAGAAUCCGCGC 22 rCEBPra PR2CCAGGAACUCGUCGUUGAA 23 UUCAACGACGAGUUCCUGG 24 PR1 GAGCUUUGGGCCCGUAAGA 25UCUUACGGGCCCAAAGCUC 26 PR2 GGUGGAUACGUUAAAGAGU 27 ACUCUUUAACGUAUCCACC 28PR3 CCCAGAAUGCCUGUGAUCA 29 UGAUCACAGGCAUUCUGGG 30 human PR4CCGAUGUUCAGUUAUCAAU 31 AUUGAUAACUGAACAUCGG 32 HNF4A BC1GAAGAUUGCUCGUGCAAAU 33 AUUUGCACGAGCAAUCUUC 34 BC2 CAGAUAUGCUCCAGUGAUG 35CAUCACUGGAGCAUAUCUG 36

TABLE 2 SEQ SEQ ID ID ID Target (Sense) NO Anti-sense (Guide) NO MousePR1 GAAAGACUCGCUCUAAUAU 37 AUAUUAGAGCGAGUCUUUC 38 albumin PR2CUAUGAGACCGUAAUAAAU 39 AUUUAUUACGGUCUCAUAG 40 PR3 CCAUUAUUGUCAUCAAAGA 41UCUUUGAUGACAAUAAUGG 42 PR4 AAGUUAGAAUCUUCCAUAA 43 UUAUGGAAGAUUCUAACUU 44

EXAMPLES Example 1 Designing Short RNAs for Upregulating AlbuminExpression

The gene sequences of genes involved in albumin production were selectedfor designing short activating RNA molecules for its specificactivation. Particularly suitable are the albumin gene, the CEBPA geneand/or the HNF4α gene.

Four parameters were used: 1) targeting gene annotations from UCSCRefSeq database; 2) targeted sequence from antisense RNA; 3) promoterselection of antisense sequences; and 4) identification of candidateshort activating RNAs.

First, the method downloads information about the target's genomiclocation, orientation, and transcriptional structure from availabledatabases (RefSeq at UCSC). Second, given a database of RNA transcriptswith known read direction, such as the UCSC Spliced EST track, ourmethod searches the database for transcripts that are antisense to andin the vicinity of the target gene. More specifically, the methodidentifies antisense transcripts that (a) overlap the target's promoterand the target mRNA's 5′ end; (b) overlap the target mRNA; (c) are atmost 20-100 kb upstream of the target's transcription start site (TSS);or (d) are at most 20-100 kb downstream of the target's poly-adenylationsite. The method uses these four criteria as hierarchical filters suchthat if it finds antisense transcripts that for example satisfycriterion (a), the method does not consider the three other criteria.Third, based on the target's TSS, the method downloads the antisensegenomic sequence from a fixed size region upstream and downstream of theTSS. The typical region size used by the method is 500 nts upstream anddownstream of TSS, but larger or smaller sizes can also be used. Fourth,the method designs siRNAs that give effective and specificdown-regulation of the antisense target sequence. The method (a) uses asiRNA design algorithm, such as GPboost (Seatrom P, 2004), to identifycandidate effective siRNAs; (b) removes all candidate siRNAs with aaaa,cccc, gggg, or uuuu motifs and GC content less than 20% or greater than55%; (c) removes all candidates that have Hamming distance less than twoto all potential off-target transcripts; and (d) returns a given numberof remaining non-overlapping siRNAs sorted by their predicted siRNAknockdown efficacy. The method returns the two highest scoring saRNAsfor a given antisense target sequence.

Following a denaturation step at 90° C., paired saRNA oligonucleotideswere annealed using 50 mM Tris-HCl, pH 8.0, 100 mM NaCl and 5 mM EDTA.

Example 2 Upregulation of Albumin Expression through Transfection withAlbumin saRNA

Materials and Methods

25 nM of annealed Albumin saRNA designed as described in Example 1 wastransfected onto a monolayer of cell using Nanofectamine (PAA, UK)following the manufacturer's instructions. This process was repeatedthree times. The sequences of the saRNA that were used for this studywere human albumin PR1 (SEQ ID NO:5 and SEQ ID NO:6), human albumin PR2(SEQ ID NO:7 and SEQ ID NO:8), human albumin PR3 (SEQ ID NO:9 and SEQ IDNO:10) and human albumin PR4 (SEQ ID NO:11 and SEQ ID NO:12), as shownin Table 1. A random, scrambled RNA molecule was used as control.

After transfection, the isolation of total RNA was performed using theRNAqueous-Micro kit (Ambion, UK) following the manufacturer'sinstructions. Briefly, the cells were gently centrifuged followed by 3pulses of sonication at Output 3 in Lysis buffer (Ambion, UK). The celllysates were then processed through an RNA binding column, followed bymultiple washes and elution. The total RNA isolated was quantified by aNanodrop 2000 spectrophotometer. 500 ng of total extracted RNA wasprocessed for elimination of genomic DNA followed by reversetranscription using the QuantiTect® Reverse Transcription kit fromQiagen.

The isolated RNA extracts were analysed using quantitative reversetranscriptase (qRT-PCR). Briefly, the extracts were reverse transcribedusing First strand cDNA synthesis kit (Qiagen). The cDNA was thenamplified for quantitative analysis using QuantiFast® SYBR®Green PCR Kitfrom Qiagen. Amplification was performed using Applied Biosystems 7900HTFAST-Real-Time System with 40 cycle conditions at 95° C. for 15 secondsand 60° C. for 45 seconds with a total volume of 25 μl per sample.Amplified products were then analysed using Applied Biosystems RQManager 1.2.1. 5 independent experiments were amplified in triplicatesfor quantitative analysis. Student T-Test scoring was performed at 99%confidence intervals.

Albumin production was determined within the cells through the use of analbumin ELISA. Briefly, the cells were grown in phenol-red free RPMImedia in the presence of charcoal stripped FCS. Following three sets ofsaRNA transfections at 8 hrs, 16 hrs and 24 hrs, the culture media wascollected for total Albumin ELISA (Assay Max, Albumin ELISA, Assay ProUSA) following the manufacturer's instructions.

Results

The effects of the transfection with the albumin saRNA oligonucleotideson albumin production are illustrated in FIG. 2. An RT-PCR profile ofthe mRNA levels showed an increase in albumin levels only in cellstransfected with saRNAs.

FIG. 10A-10C show that a significant increase in the albumin mRNA isdetected in the transfected HepG2 cell line (FIG. 10B) and rat liverepithelial cells (FIG. 10C) as compared to control, and this in turnleads to a significant increase in albumin production in the transfectedcell lines (FIG. 10A), data only shown for HepG2).

Example 3 Inhibition of the Proliferation of Cells Through Transfectionwith Albumin saRNA

HepG2 cells and rat liver epithelial cells were transfected with albuminsaRNAs as described in Example 2, i.e. the same saRNAs were used in thisExample as in Example 2. Cell proliferation within the rat liverepithelial cells and HepG2 cells respectively was measured using theWST-1 proliferation assay. Briefly, the cell proliferation reagenttetrazolium salt,4-[3-(4-iodophenyl)2-(4nitrophenyl)2H-5-tetrazolio]1,3benzenedisulfonate (Roche Applied Science, UK) was added (e.g., following themanufacturer's instructions). The colorimetric assay was then incubatedfor 30 minutes to allow cleavage of the tetrazolium salt WST-1 to aformazan dye by mitochondrial succinate-tetrazolium reductase in viablecells. The quantity of formazan dye, which is directly related to thenumber of metabolically active cells proliferating was measured atAmax450 nm in a multiwall plate reader.

Cell proliferation and viability was significantly inhibited by each ofthe saRNAs. Results obtained with the HepG2 cell line are shown in FIG.3.

Example 4 Upregulation of Albumin and Inhibition of Cell Proliferationthrough the Transfection with CEBPA saRNA In Vitro

The following saRNA duplexes (sense/antisense) targeted for CEBPA wasused for this study:

AW1 Sense strand:  (SEQ ID NO: 13) CGGUCAUUGUCACUGGUCAAW1 anti-sense strand:  (SEQ ID NO: 14) UGACCAGUGACAAUGACCGAW2 Sense strand:  (SEQ ID NO: 15) AGCUGAAAGGAUUCAUCCUAW2 anti-sense strand:  (SEQ ID NO: 16) AGGAUGAAUCCUUUCAGCU

Synthetic saRNA duplexes (above) targeting the 3′UTR promoter region ofthe CEBPA was transfected into either cell lines or primary CD34+ cells(Omnicytes) using the liposomal method (Nanofectin). Changes in thetranscript levels of CEBPA and albumin was measured quantitatively byqPCR. Additionally changes in cellular proliferation was measured usingtetrazolium salt,4-[3-(4-iodophenyl)2-(4nitrophenyl)2H-5-tetrazolio]1,3benzenedisulfonate (WST-1) assay.

Since the expression of CEBPA in HepG2 cells is lower when compared toother cultured human hepatocytes, HepG2 cells were used to investigatethe effects of transfecting AW1 and AW2. Following three doses oftransfection over a culture period of 48 hours, HepG2 cells wereharvested and analysed for mRNA analysis. No significant changes in themRNA levels of CEBPA was observed during this culture period (FIG. 11A).This is unsurprising as CEBPA shows early and fast decay during initialstages of culture. In contrast, an increase in mRNA transcript levels ofalbumin was observed (FIG. 11B). To confirm if albumin increase in mRNAalso reflected its translation to protein, a functional enzyme-linkedimmunosorbent assay (ELISA) was performed. saRNA transfected cells werecultured in a serum free/charcoal stripped media to remove any source ofexogenous albumin. Following the 48 hour culture period in the presenceof either AW1 or AW2 the cell culture medium was isolated and processedfor detection of human albumin using a commercially available kit. Asignificant increase of albumin expression was detected in cellstransfected in saRNA when compared to untransfected cells (FIG. 11C). Wealso performed a WST-1 proliferation assay in the transfected HepG2cells. A reduction in cell proliferation was observed (FIG. 11D).

We next determined if AW1 or AW2 would successfully induce a positiveregulation of albumin transcript or translation in non-hepatic cells,namely a prostate cancer epithelial cell line (DU145) (FIG. 12A-12C) anda healthy adult haematopoietic CD34 cell line. These cell lines weretransfected with AW1 and AW2 as described above, following three dosesover a period of 48 hours. The cells were then harvested and analysedfor transcript levels of CEBPA and albumin. The prostate cancer cellline showed a strong increase in CEBPA transcript level when transfectedwith AW2 relative to AW1 (FIG. 12A) and both transfections induced asignificant increase in albumin transcript levels (FIG. 12B). A WST1assay was also performed. Growth of DU145 was significantly reducedfollowing transfection with CEBPA (FIG. 12C).

AW1 and AW2 also upregulated albumin production in the CD34+ stem cells,but the proliferation of the CD34 cells was not affected by the the AW1and AW2 saRNA constructs (data not shown).

The inventor has successfully established that transfection with saRNAAW1 and AW2 specific to CEBPA has the ability of up-regulating albumintranscript level and protein expression in a human hepatocellularcarcinoma line and a prostate cancer epithelial cell line. Furthermore,this increase in albumin protein expression leads to a decrease in cellproliferation. By contrast, the transfection of healthy CD34 cells withsaRNA AW1 and AW2 specific to CEBPA did not affect cell proliferation,suggesting that the effect on cell proliferation is specific to cancercells.

Example 5 Upregulation of Albumin Through the Transfection with AlbuminsaRNA in Mouse Liver In Vivo

Ten Male C57B16/J, 8 week old mice were used for the experiment (controlgroup N=5). Approval was obtained from Institutional and RegionalRegulatory bodies and all procedures were in compliance with standingNational Regulations.

Albumin saRNA oligonucleotides were developed as described in Example 1.Mouse albumin PR2 (SEQ ID NO:39 and SEQ ID NO:40), as shown in Table 2,was chosen for this study. The oligonucleotides were reconstituted with100 μL of RNase/Dnase free H₂O; 50 μL of complex A and 50 μL of complexB (InvivoFectamine, Invitrogen, CA, USA) were mixed, incubated at 50° C.for 30 minutes and were used for tail vein injections. Control animalswere injected with equal volume of PBS while a positive control animalreceived siRNA against Factor 7; a total of 5 control and 5 experimentalanimals were injected.

After administration, the total RNA was isolated. Frozen tissue sectionswere placed into scintillation vials containing Trizol and homogenisedfor 30 seconds. The homogenate was then transferred in Falcon tubes fora further 2 minutes of homogenisation. Chloroform was then added to thisand mixed by vortexing followed by a centrifugation step at 12,000 rpmfor 15 minutes at 4° C. The aqueous upper phase was then transferredinto a fresh microfuge tube where RNA was precipitated using 5 mg/ml oflinear acrylamide (Ambion) and isopropanol overnight at −20° C. The RNAwas pelleted by centrifugation at 12,000 rpm for 15 minutes at 4° C. andwashed with ice cold 70% ethanol. The RNA was pelleted again at 7,500rpm for 5 minutes at 4° C. The supernatant was removed immediately andthe RNA pellet allowed to air dry. The RNA was dissolved in nucleasefree water for immediate analysis for RNA integrity using aBionanalyser.

The isolated RNA was analysed using qRT-PCR as described in Example 2.Albumin production was determined using an Albumin ELISA as described inExample 2.

FIG. 13A shows that the administration of albumin saRNA oligonucleotidesusing a dendrimer delivery vehicle to a mouse leads to a significantincrease in albumin within the blood circulation.

The administration of the albumin saRNA had no dentrimental effects onoverall liver function according to the liver function markers gammaglutamyl transpeptidase (FIG. 13B), alanine aminotransferase (FIG. 13C)and aspartate aminotransferase (FIG. 13D) or bilirubin (data not shown).Furthermore, the albumin saRNA was able to downregulate the mRNAexpression of the genes which encode a fetoprotein (FIG. 16A) andhepatocyte growth factor (FIG. 16B). As both of these proteins arelinked to hepatocyte proliferation, the downregulation of these genes bythe saRNA suggests that they are capable of inhibiting proliferation invivo.

The immunohistochemistry of the mice livers showed that the architectureof the liver acini was preserved, there was no significant portalinflammation or fibrosis, the bile ducts, central venules and thesinusoids were unremarkable, there were not foci of oval cellproliferation, there were no distinct foci of hepatiticnecroinflammatory activity, there was not activation of Kuppfer cells,at least not one detectable by morphology, there were no vascular orendothelial alterations, there were not signs of reversible cell injury,i.e. ballooning or steatosis and there were no findings suggestive ofincreased hepatocellular proliferation, i.e. mitoses, thickened plates,nuclear crowding. In summary, the morphology of the liver remainedmostly unchanged by the administration of the albumin saRNAoligonucleotides.

Example 6 Upregulation of Serum Albumin Through the Transfection withCEBPA saRNA in Rats with Cirrhotic Livers

The ability of the CEBPA saRNA constructs to increase albumin wasassessed on diseased animals, namely rats with cirrhotic livers.

In order to assess the in vivo effects of AW1 (SEQ ID NO: 13 and SEQ IDNO:14) and AW2 (SEQ ID NO:15 and SEQ ID NO:16) on albumin production,rats with cirrhotic livers were administered with AW1 and AW2 constructsand albumin ELISA was used as described in Example 2.

The administration of the AW1 construct led to a significant increase inserum albumin (p=0.0288) (FIG. 14). Pooling the AW1 and the AW2 datatogether shows that the increase in albumin production is even moresignificant (p=0.0172) (FIG. 15), wherein AW1 and AW2 pooled arelabelled “CEBPA”). This shows that effects of the CEBPA saRNA constructson albumin production can be seen in diseased animals in vivo.

Example 7 Inhibition of Tumor Development and Growth ThroughTransfection with CEBPA saRNAs in a Rat Tumor Model

20 rats were treated with carbon tetra chloride (CCl4) to induce livercirrhosis. The rats were treated with 0.2 mL/100 g body weight of CCl4at concentration of 40 mL/L twice a week for 4 weeks.

Then they were randomised into two groups. A control group was injectedwith saline in the tail vein. The experimental group injected with threeinjections of saRNA which upregulates albumin by upregulating CEBPA: AW1(SEQ ID NO: 13 and SEQ ID NO:14) or AW2 (SEQ ID NO:15 and SEQ ID NO:16)at day 1, 3 and 5. All animals were sacrificed two weeks after the saRNAinjection.

The rats treated with saRNA rats had a significantly smaller number oftumours, and the tumours were smaller compared to control(saline-treated) rats. Moreover, the onset of tumour developments waslater in the saRNA treated group. AW1 was particularly effective atinhibiting tumour development and growth.

Example 8 Inhibition of Tumor Development and Growth ThroughTransfection with Albumin-Upregulating saRNAs in an Animal Tumor Model

The experiment described in Example 6 or 7 is repeated with mice whichhave been chemically induced to have liver cancer. Liver cancer isinduced using DEN, a genotoxic carcinogen. DEN is typically administeredto mice between 12 and 15 days of age by a single intraperitonealinjection (5 μg/g body weight). Using this protocol, 100% of B6C3F1 malemice develop HCCs, on average, 44 wk after intraperitoneal injection ofDEN. saRNAs shown in Table 1 are then administered and albuminexpression is assayed as described in Example 6.

Tumor diameters are measured with digital calipers, and the tumor volumein mm³ is calculated by the formula: Volume=(width)²×length/2. Tumordevelopment and growth is analysed by determining tumor volume oftreated mice compared to control mice.

Example 9 Inhibition of Cancer Cell Proliferation Through theTransfection with Albumin-Upregulating saRNAs in Mice with a HumanTumour Xenograft

Human liver tumour cells are cultured in vitro, washed, and injected(3.0×10⁶ cells) subcutaneously into the lower flank of nude mice (4-6weeks old). Therapy using saRNAs shown in Table 1 is started after 1-3weeks when the tumours have reached an average volume of ˜50-60 mm³.Tumour diameters are measured with digital calipers, and the tumourvolume in mm³ is calculated by the formula: Volume=(width)²×length/2.

saRNA administration and albumin expression assays are carried out asdescribed in Example 6. Tumour development and growth is analysed bydetermining tumour volume of treated mice compared to control mice.

I claim:
 1. A method of treating or preventing a hyperproliferativedisorder and/or a disorder characterised by hypoalbuminemia, said methodcomprising administering to a subject in need thereof a short activatingRNA which up-regulates albumin production, wherein said short activatingRNA upregulates albumin production by activating a target gene, whereinthe target gene is albumin.
 2. The method according to claim 1, whereinsaid hyperproliferative disorder is liver cancer or prostate cancer. 3.The method according to claim 1, wherein said disorder characterised byhypoalbuminemia is cirrhosis, hepatitis or oedema.
 4. The methodaccording to claim 1, wherein said short activating RNA activatesalbumin by specifically targeting a target RNA transcript, (i) saidtarget RNA transcript is complementary to a sequence located on thecoding strand of the target gene between 500 nucleotides upstream and500 nucleotides downstream of the transcription start site of the targetgene, and (ii) said short activating RNA comprises a sequence of atleast 18 nucleotides which has at least 95% complementarity to a regionof the target RNA transcript.
 5. The method according to claim 1,wherein said short activating RNA molecule is a single or doublestranded RNA molecule up to 30 nucleotides in length.
 6. The methodaccording to claim 1, wherein said short activating RNA comprises afirst strand having a sequence selected from SEQ ID NOs: 6, 8, 10, 12,38, 40, 42, and
 44. 7. The method according to claim 6, wherein saidshort activating RNA comprises a second strand having a sequenceselected from SEQ ID NOs: 5, 7, 9, 11, 37, 39, 41, and
 43. 8. The methodaccording to claim 5, wherein said short activating RNA is a doublestranded RNA molecule and comprises a number of unpaired nucleotides atthe 3′ end of each strand forming 3′ overhangs.
 9. The method accordingto claim 8, wherein said 3′ overhang is UU or UUU.
 10. The methodaccording to claim 1, wherein the short activating RNA inhibits cellproliferation.
 11. A method according to claim 10, wherein said cell isa hyperproliferative cell.